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Nanospanlastics as a Novel Approach for Improving the Oral Delivery of Resveratrol in Lipopolysaccharide-Induced Endotoxicity in Mice

Nanospanlastics as a Novel Approach for Improving the Oral Delivery of Resveratrol in... Purpose Resveratrol (RSV) is a natural polyphenolic compound that has numerous biological effects. Owing to its poor bioavailability, only trace concentrations of RSV could be found at the site of action. Therefore, the present study was aimed at developing RSV-loaded nanospanlastics to improve its oral delivery and therapeutic activity. Methods RSV-loaded nanospanlastics were prepared using the thin film hydration technique. The developed formulations were characterized via vesicular size (VS), polydispersity index (PDI), zeta potential (ZP) measurements, fourier transform infrared (FT-IR) spectroscopy analysis and transmission electron microscopy (TEM). In vitro release profile was carried out using dialysis bag diffusion technique. In vivo study was carried out using lipopolysaccharide (LPS)-induced endotoxicity model in mice to evaluate the formulations activity. Results The results revealed the successful development of RSV-loaded nanospanlastics which exhibited EE% ranging from 45 to 85%, particle sizes ranging from 260.5 to 794.3 nm; negatively charged zeta potential (≤ − 20 mV) and TEM revealed their spherical shape. An in vitro release study showed biphasic pattern with sustained release of drug up to 24 h. In vivo results showed the superiority of RSV-loaded nanospanlastics over conventional niosomes in attenuating serum levels of liver and kidney functions (aspartate transaminase (AST), alanine transaminase (ALT), and creatinine) in LPS-induced endotoxic mice. Furthermore, both of them suppressed the elevated oxidative stress and inflammatory markers (malondi- aldehyde (MDA), nitric oxide (NO), and interleukin-1beta (IL-1β)) estimated in the liver and kidney tissues. However, the nanospanlastics showed a prevalence effect over conventional niosomes in kidney measurements and the histopathological examinations. Conclusions These findings reveal the potential of nanospanlastics in improving the oral delivery and therapeutic efficacy of RSV. Keywords Resveratrol · Nanospanlastics · Niosomes · Lipopolysaccharide · Endotoxicity Introduction that harm the host via triggering a systemic inflammatory response with the onset of septic shock and may lead to Systemic inflammation is considered a hallmark of sepsis death [2]. Upon activation of macrophages by LPS, the that can be provoked by parasites, bacteria, mycobacteria, ina fl mmatory response is initiated by a cascade of mediators viruses, and fungus but mainly by gram-negative bacteria including cytokines, mainly interleukins and tumor necrosis [1]. Lipopolysaccharide (LPS) is an endotoxin constitu- factor-α, as well as the production of reactive oxygen spe- ent derived from the cell walls of gram-negative bacteria cies (ROS) and reactive nitrogen species (RNS), followed by alteration of hemostasis that finally led to multiple organ dysfunction [3, 4]. * Mostafa Mohamed Younis Resveratrol (RSV) is a natural polyphenolic compound most_younis@hotmail.com formed by plants as a result of abiotic stresses, like ultraviolet Pharmaceutical Technology Department, National Research (UV) light exposure or existence of heavy metal ions. RSV can Centre, El-Buhouth Street Dokki, Cairo, Egypt be found in grapes, nuts, and blackberries. It is considered a Drug Radiation Research Department, National Centre novel nutraceutical, providing consumers with a wide range of for Radiation Research and Technology (NCRRT), Egyptian health benefits in addition to its basic nutritional value. RSV Atomic Energy Authority, Nasr City, Cairo, Egypt Vol.:(0123456789) 1 3 Journal of Pharmaceutical Innovation exhibits geometric isomerism, but trans-resveratrol (trans-3,4,5- safe deformable nanovesicles. Both hydrophilic and hydrophobic trihidroxystilbene) is the only isomer that shows biological activ- drugs can be delivered via nanospanlastics, where these drugs ities such as anticancer, anti-inflammatory, antidiabetic, cardio- are enclosed in the inside hydrophilic compartment and the outer protective, and antiaging [5]. The protective effect of RSV in the lipid layer, respectively [30]. Nanospanlastics differ from conven- LPS-induced endotoxemia model has been previously reported, tional niosomes in terms of their structural rigidity. Cholesterol as RSV was able to hinder the induced oxidative stress in the is known to increase the rigidity of the niosomal structure and brain, liver, and kidney [6–8] and to interfere with the inflam- make the vesicles less elastic [27]. However, the inclusion of an matory signaling cascades in such model [9]. Despite the fact EA in nanospanlastics formulations offers significant flexibility that RSV was therapeutically used in experimental models of because the size and zeta potential of the formulations can be inflammation, its limited bioavailability is regarded a limiting changed to meet specific requirements using simple and reliable factor. Combination therapy was one of the widely investigated procedures [31]. Moreover, they are more chemically stable than methods to overcome bioavailability issues such as combination conventional niosomes [32]. with bioenhancing agent or low-dose radiation [10–12]. Researchers have previously studied spanlastics as a vital Due to poor bioavailability of RSV, as it exhibits rapid drug delivery system via topical route [25, 33]. One of the metabolization and excretion, only minute amounts of free goals of the present study is to develop RSV-loaded nanospan- RSV (below 5 µg/ml) can be found in systemic circulation lastics and investigate spanlastics as an oral drug delivery sys- after 25 mg oral dose [13, 14]. Also, RSV is poorly water tem. Thereafter, employing the RSV loaded nanospanlastics soluble with a log p value of 3.1, and according to the Euro- therapeutically via oral route in LPS-induced endotoxemia pean Pharmacopeia, it is considered “practically insoluble in model, aiming to improve its anti-inflammatory efficacy. water”[15]. In addition, RSV has a short half-life of about 8–14 min [16]. Hence, there is limitation in the clinical appli- cation of RSV, which suggests the development of novel for- Materials and Methods mulations to protect and stabilize RSV from being degraded, improve its water solubility, provide a prolonged release, Materials enhance its bioavailability, and target it to definite sites [17]. It is vital to overcome RSV’s poor water solubility and Chemicals limited bioavailability in order to clinically translate its posi- tive effects. Efforts to increase the efficiency and safety of Trans-resveratrol (purity ≥ 98%) was purchased from Carl- therapeutic drugs have focused on integrating the medica- Roth (Karlsruhe, German). Lipopolysaccharide (LPS from tions into a nanocarrier [18, 19]. Nanosized drug delivery E. coli serotype 055:B5) was procured from Sigma-Aldrich systems have established special consideration aiming at (St. Louis, MO, USA). Sorbitan monostearate (Span 60) and reducing the adverse effects and enhancing the drug ther - Sorbitan monooleate (Span 80) were procured from Merck apy efficiency [20, 21]. To improve in-vivo delivery of (Schuchardt OHG, Germany). Sorbitan monopalmitate (Span RSV, various nanoparticle (NP)-based formulations have 40) and cholesterol were purchased from Sigma-Aldrich (St. been studied; these studies comprise drug encapsulation Louis, MO, USA). Tween 80 was obtained from Loba Chemie into conventional colloidal carriers such as liposomes [22], Co. (India). Kits employed for the measurement of aspartate loading into solid lipid NPs [23], and encapsulation into transaminase (AST), alanine transaminase (ALT), malondi- biodegradable polymeric NPs [24]. Yet, these nanosystems aldehyde (MDA), and nitric oxide (NO) were obtained from exhibit rigid nature that lacks deformability and flexibility Biodiagnostic (Cairo, Egypt). Enzyme-linked immunosorbent throughout their way across the biological membranes [25]. assay (ELISA) kit for the measurement of interleukin-1beta Thus, more investigations have been recently performed to (IL-1β) was purchased Abcam (Cambridge, MA, USA). Other improve their elasticity in order to increase their permeabil- used chemicals were of analytical grade. ity through different biological membranes [26]. Nanospanlastics are surfactant-based nanovesicular carriers Animals that were first set up by Kakkar and Kaur [27]. Nonionic sur - factants and edge activators are the main constituents of nano- Male albino Swiss mice weighing 25–30 g were sourced spanlastics [15]. They have a good compatibility with biological from the animal breeding facility of the National Centre for systems and a low toxicity owing to the inclusion of nonionic Radiation Research and Technology (NCRRT). Animals surfactants in their structure [28]. Edge activators act by enhanc- were acclimatized for at least one week before experiment ing the permeability and flexibility of the nanocarriers’ vesicu- in the animal facility of NCRRT. They were allowed to feed lar membranes through the biological membranes by squeezing on laboratory chow and water ad libitum. All animal experi- through different pores of the biological layers without disruption ments complied with the Animal Research Reporting of In- [29]. Nanospanlastics are biodegradable, nonimmunogenic, and Vivo Experiments (ARRIVE) guidelines and were carried 1 3 Journal of Pharmaceutical Innovation out in accordance  with the National Research Council’s of Korea). The pellet was then resuspended in 10 ml distilled Guide for the Care and Use of Laboratory Animals (NIH water till further investigation. Drug amount entrapped in the publications No. 8023, revised 1978). The in vivo study was developed formulation was detected by lysis of 1 ml of resus- performed according to the guidelines set by the Research pended pellet after addition of adequate quantity of ethanol Ethics Committee at the NCRRT (permit number: 57 A/ 21). and sonication for 5 min in a bath sonicator. The content of free RSV was then estimated employing a UV–Vis spec- Methods trophotometer (Shimadzu UV–Visible spectrophotometer, 2401/PC, Tokyo, Japan) at 305 nm. Drug entrapped amount Preparation of RSV‑Loaded Nanospanlastics was calculated, as entrapment efficiency percentage (EE%), in triplicate, according to the following equation [37]: Drug-loaded nanospanlastics were developed by the thin film Amount of entrapped drug hydration technique [26, 34] employing nonionic surfactants EE% = × 100 Total drug added (NIS) such as Span 60 or Span 40 along with cholesterol. Two edge activators (EA), namely, Span 80 or Tween 80, were used at NIS: EA molar ratios of 9:1 or 8:2. Absolute ethanol and Determination of Vesicular Size (VS), Polydispersity chloroform were used in the ratio (1:1 v/v) as a solvent to dis- Index (PDI), and Zeta Potential (ZP) Measurements solve the drug [29]. In brief, one of the selected NIS, with the edge activators, in definite molar ratios, together with 10 mg VS, PDI, and ZP of the prepared nanospanlastics and con- drug was dissolved in the organic mixture in a round bottom ventional niosomes were measured by dynamic light scatter- flask [35, 36]. The mixture of solvents was then evaporated ing (DLS) using Malvern Instruments (Malvern, UK; Nano under reduced pressure by a rotary evaporator (Rotavapor, ZS). Multimodal mode was employed in all measurements. Buchi-M/HB-140, Switzerland) to form a thin film on the ZP measurements were determined after injection of the wall of the flask. This lipid film was then hydrated in 10 ml diluted sample into the specified cell. The measurements phosphate buffer pH 7.4, kept at 60 °C, under rotation for 1 h were done inFig. 4a–c) triplicate. [37]. Conventional niosomes were also prepared for compari- son using the same method where the same steps took place but without adding edge activators. The composition of RSV- In Vitro Release Profiles loaded vesicular systems is shown in Table 1. Drug release from RSV suspension, as well as from the Characterization of RSV‑Loaded Nanospanlastics selected RSV-loaded nanospanlastics and RSV-loaded niosomes, was investigated by dialysis bag diffusion tech- Drug Entrapment Assessment nique employing a shaking water bath (Memmert, SV 1422, Schwabach, Germany). In a cellulose dialysis bag (dialysis The prepared RSV-loaded nanospanlastics were separated by tubing cellulose membrane, Sigma Co., USA; Molecular centrifugation at 8000 rpm at 4 °C for 45 min using cooling weight cutoff 12,000–14,000), an aliquot of the resuspended centrifuge (Union 32R, Hanil Co., Gyeonggi-do, Republic RSV-loaded nanospanlastics (equivalent to 2 mg RSV) was Table 1 Composition, entrapment efficiency and physicochemical properties of RSV-loaded nanospanlastics/niosomes Code Molar ratio (w/w %) EE VS PDI ZP (%) ± S.D (nm) ± S.D (mV) ± S.D Span 60 Span 40 Span 80 Tween 80 CHOL F1 9 – – 1 – 62.63 ± 0.36 794.3 ± 97.78 0.411 − 33.1 ± 5.71 F2 8 – – 2 – 49.4 ± 0.03 618.2 ± 73.30 0.371 − 30.5 ± 7.26 F3 9 – 1 – – 76.31 ± 0.04 716.2 ± 74.29 0.407 − 35.8 ± 3.04 F4 8 – 2 – – 68.96 ± 0.14 558.1 ± 77.54 0.237 − 38.9 ± 6.95 F5 1 – – – 1 85.03 ± 0.02 328.8 ± 58.10 0.339 − 21.3 ± 7.04 F6 – 9 – 1 – 55.42 ± 0.03 398.5 ± 70.64 0.316 − 33 ± 4.12 F7 – 8 – 2 – 45.84 ± 0.03 357.7 ± 47.29 0.304 − 35.7 ± 6.27 F8 – 9 1 – – 65.3 ± 0.04 361.6 ± 50.14 0.355 − 41.9 ± 6.11 F9 – 8 2 – – 62.79 ± 0.05 289.5 ± 71.90 0.432 − 34.3 ± 7.19 F10 – 1 – – 1 71.28 ± 0.09 260.5 ± 52.17 0.388 − 46.2 ± 3.60 Data are expressed as the mean ± S.D. (n = 3) 1 3 Journal of Pharmaceutical Innovation added. Both ends of the bags were sealed. Based on solubil- In Vivo Studies ity characteristics of resveratrol and to fulfill sink condition, they were placed in a release solution (media) containing Experimental Design 100 ml of phosphate buffer pH 7.4 and 30% ethanol to reach sink condition [38, 39]. The shaking water bath was set at a LPS-induced endotoxicity model was carried out in the in-vivo temperature of 37 ± 0.5 °C and a rotational speed of 100 rpm. studies to evaluate the anti-inflammatory efficacy of selected At different time intervals (1, 2, 3, 4, 5, 6, 8, and 24 h), sam- RSV formulations. Mice were divided into six groups (n = 6). ples were withdrawn and substituted with same volumes of In Groups I and II, mice received saline and served as negative fresh medium to maintain sink condition. Drug concentra- and positive controls, respectively. In Group III, mice received tions were then determined in the withdrawn samples spec- free RSV (50 mg/kg) suspended in saline [3, 12]. In Groups trophotometrically at 305 nm. The ratio of RSV released to IV and V, mice received RSV-loaded nanospanlastics (F8) the total amount in the dialysis bag was used to calculate the and RSV-loaded niosome (F10) formulations, respectively. cumulative percent of RSV released. All measurements were The dose of RSV in F8 and F10 formulations is equivalent carried out three times. to that of the free drug group. In Group VI, mice received In vitro release results were subjected to various math- dexamethasone (DEX; 2 mg/kg) and served as the standard ematical models such as zero-order, first-order, Higuchi, and anti-inflammatory drug group [43]. Peppas to study the release mechanism of RSV from the Treatments were orally administered daily for 3 consecutive prepared nanosystems. The linear regression analysis for days except Group VI which was injected intraperitoneally with the release data was assessed by Microsoft Excel Program. dexamethasone only once at the third day [3, 9]. After 1 hour The mechanism was determined using the regression coef- from the last treatment dosage, a single dose of LPS (1 mg/kg) 2 2 ficient (R ). R close to 1 was considered to be the best fit was injected intraperitoneally into all animals [44, 45] except model. The exponent “n” was determined for Peppa’s model for Group I, which served as the normal/negative control group. to determine the RSV release mechanism. According to The current dose of RSV was selected according to the previ- Peppa’s theory, if n ≤ 0.43, the drug is released via a Fick- ous study of El-Ghazaly et al. [12] as they postulated that RSV ian diffusion mechanism, if 0.43 < n < 0.85, the mechanism exhibits a submaximal anti-inflammatory effect at such dose is non-Fickian diffusion, if n = 0.85, the mechanism is case level in the carrageenan-induced acute inflammation model. II transport, and if n > 0.85, the mechanism is super-case II transport [40, 41]. Sampling Procedure Fourier Transform Infrared (FT‑IR) Spectroscopy Analysis At the end of experiment, mice were sacrificed by decapita- tion under urethane anesthesia, 5 hours post LPS injection. FT-IR spectroscopy analysis was performed for the selected The blood samples were collected, and serum was obtained formulations F8 and F10, along with individual compo- by cooling centrifugation at 4000 rpm at 4 °C for 10 min and nents (RSV, and Span 40) on a JASCO 6100 FT-IR spec- then divided into small aliquots that were stored at − 80 °C for trophotometer (JASCO, Tokyo, Japan). Potassium bromide eventual use in estimating liver and kidney functions. There- (KBr) was added to the sample first. The mixture was then after, livers and kidneys were dissected, washed with saline, compacted for 2 min using a hydraulic press at a pressure and homogenized in ice-cold PBS to prepare 20% homogen- of 200  kg/cm . At wavenumbers ranging from 4000 to ates which were centrifuged at 4000 rpm at 4 °C for 10 min. −1 400  cm , a KBr pellet manufactured for each sample was The supernatants were then distributed into several aliquots and scanned against a KBr blank pellet. stored at − 80 °C to be employed for biochemical tissue analy- sis. Parts of the livers and kidneys from each group were kept Transmission Electron Microscopy (TEM) in 10% formalin, to be used for histopathological evaluation. TEM was performed to determine the morphological charac- Evaluation of Liver and Kidney Functions teristics of the selected vesicles [42]. Samples were diluted ten folds with bidistilled water and mixed well prior to the exami- The liver and kidney functions (ALT, AST, and creatinine) were nation. A drop of the investigated formulation was put on a measured in serum via a colorimetric method using commer- carbon-coated copper grid. Then, it was air-dried at room tem- cially available kits according to the manufacturer’s instruc- perature for 10 min. Afterwards, phosphotungstic acid solution tions. The absorbance of each sample was measured using the (1% (w/v)) was dropped to the grid and dried. The grid was then Unicam 8625 UV/V spectrophotometer (Cambridge, UK). loaded to TEM (JEOL Co., JEM-2100, Tokyo, Japan). 1 3 Journal of Pharmaceutical Innovation 85.03 ± 0.022%. The results  reflect the effects of nonionic Evaluation of Oxidative Stress and Inflammatory Markers surfactant type (Span 60 and Span 40) and edge activators (Span 80 and Tween 80) on EE% for all prepared formula- Liver and kidney contents of MDA and NO were estimated tions. It could be concluded that nanospanlastics prepared using Span 60 showed higher EE% than those prepared by a colorimetric method using commercially available kits, and the absorbance of each sample was measured using a using Span 40 at the same molar ratio and same edge acti- vator. This may be attributed to the effect of the surfactant spectrophotometer, while the interleukin-1beta (IL-1β) con- tent was assessed by ELISA using commercially available alkyl chain length, which is directly proportional to the EE% [47]. Span 60 has a longer alkyl chain (C16) than Span 40 kit according to the manufacturer’s instructions, and ELISA plate reader Dynatec MR5000 (Guernsey, Channel Island, (C14); this resulted in a higher EE%. Moreover, HLB plays an important role in drug EE%; the higher the HLB of the UK) was used to measure the absorbance of each sample. surfactant, the lower will be the EE%. Since Span 60 HLB (4.7) is smaller than that of Span 40 (6.7), thus, higher EE% Histopathological Evaluation was revealed by Span 60 nanospanlastics [37]. The same was revealed, upon comparing EE% of the two conventional Tissue specimens of the liver and kidney were fixed in 10% niosomes prepared, where Span 60 niosomes (F5) showed neutral-buffered formalin. They were then trimmed, washed higher EE% than Span 40 niosomes (F10). and dehydrated in ascending grades of alcohol, cleared in The formulations F2 and F7 showed the least entrapment xylene, and embedded in paraffin blocks. Sections of 5 μm efficiencies, i.e., 49.4 ± 0.033 and 45.84 ± 0.039%, respec- were obtained by sledge microtome and stained with hema- tively, when compared to other prepared formulations. It toxylin and eosin (H&E) [46]. Images were captured and can be concluded that increasing the amount of Tween processed using Adobe Photoshop (version 8). Histopatho- 80 as edge activator led to formation of unstable bilayers, logical examinations were conducted in blind fashion. increased leakiness, and permeation of drugs enclosed in the vesicles. Also, inclusion of Tween 80, with high concentra- Statistical Analysis tions, reduces the viscosity of the formulation signifying less rigidity of the bilayer membrane. As the presence of unsatu- The results were presented as mean values with standard devia- ration in Tween 80’s alkyl chains increases the bending of tions (S.D.). One-way analysis of variance (ANOVA) was used the alkyl chains to a degree that can affect the tightness to compare statistical differences between groups, followed by of the developing vesicular membranes, these effects can the Tukey–Kramer multiple comparison test. GraphPad prism enhance membrane permeability and increase RSV escape 5 (Graph Pad Software Inc., San Diego, California, USA) was into the external phases [48]. On the other hand, the lack of used for statistical analysis. A p value of less than 0.05 was unsaturation in Span 80’s alkyl chains would allow for the considered significant. The figures were represented using the construction of more rigid vesicular membranes capable of Origin® software and the Microsoft Excel programs. reducing drug leakage [49]. The edge activator concentra- tion had a beneficial effect on drug EE% until a critical span: edge activator ratio of 4:1, in which lower drug EE % was obtained beyond this ratio [50]. Results and Discussion On the opposite side, the edge activator-free dispersions (conventional niosomes F5 and F10), which were prepared Preparation of RSV‑Loaded Nanospanlastics/Niosomes using Span 60 or 40 and cholesterol, showed the highest entrapment efficiency (85.03 ± 0.022 and 71.28 ± 0.093). RSV-loaded formulations were successfully prepared These observations reflect the important role of cholesterol employing thin film hydration technique and using different which resulted in an increase in the viscosity of the formu- nonionic surfactants and edge activators. Table 1 comprises lation and increased the rigidity and stability of the bilayer the components and the physicochemical properties of the membrane, preventing the leakiness and retard permeation developed RSV-loaded nanospanlastics/niosomes. of solutes enclosed in the niosomes [51]. Characterization of RSV‑Loaded Formulations Vesicular Size (VS), Polydispersity Index (PDI), and Zeta Potential (ZP) Measurements Drug Entrapment Assessment The mean VS, ZP, and PDI values of the developed dis- The EE% of all formulations is illustrated in Table  1, persions are presented in Table 1. Vesicle sizes of differ - where entrapment efficiencies ranged from 45.84 ± 0.039 to ent formulations were in the nanorange between 260.5 and 1 3 Journal of Pharmaceutical Innovation 794.3 nm. It could be depicted that VS of nanospanlastics prepared employing span 60 showed an increased size compared to those prepared using span 40. This increase is related to the increase of Span alkyl chain length, which results in an enhanced critical packing parameter and thus 80 an increase in vesicle size [52]. The results also reveal that smaller particles were formed upon increasing edge activa- 60 tor concentration. This might be attributed to the surfactant ability to reduce interfacial tension. A higher concentration of the edge activator decreased the surface tension, allowing F3 particle partition and produced smaller nanovesicles. Similar F5 results were reported previously [53, 54]. The positive influ- F8 F10 ence of the edge activators on the developed formulations Free RES can be observed by their lower aggregation tendency. The 05 10 15 20 25 PDI is used to determine the size distribution’s width. It Time (hr) was previously reported that when PDI values are < 0.5 this indicates homogeneity and narrow distribution [55, 56]. PDI Fig. 1 Release profiles of RSV from RSV-loaded nanospanlastics and values of the developed formulations ranged from 0.237 to niosomal formulae (F3, F5, F8, and F10) as well as free drug suspen- 0.432 revealing that the dispersed particles were homog- sion enously distributed. The potential stability of the colloidal system can be that was retained for 24 h. The same pattern was observed indicated by the ZP magnitude. It was reported that ZP val- ues >|20| usually indicate minor particle aggregation [57, with the nanospanlastics formulations F3 and F8, as fast release occurred in the first 8 h and about 40% to 60% of 58]. ZP values of all the prepared formulations showed highly negative values ranging between − 21.3 mv and − 46.2 the entrapped RSV was released. This was followed by a sustained and slow release that lasted for 24 h. Vesicular mv (Table 1), indicating a good stability. Negative ZP values for vesicles, prepared without adding any charge inducing sizes played a vital role in drug release, where vesicles with smaller sizes (F5 and F10) showed faster release than those agents, could be due to preferential adsorption of hydroxyl ions or adsorption of counter ions at the vesicle surface [59] having larger sizes (F3 and F8). This biphasic release pattern is consistent with previ- which are sufficiently high for electrostatic stabilization. Due to their highest EE% and suitable VS and ZP, RSV-loaded ously reported studies and seems to be a common feature of bilayer vesicles [63–65]. The initial rapid to moderate nanospanlastics F3 and F8 with molar ratio (Span 60 or 40: Span 80; 9:1) and conventional niosomes F5 and F10 of the release phase might be attributed to the diffusion of unen- trapped drug that may be adsorbed on the surface of the molar ratios Span 60 or Span 40: Cholesterol (1:1) were selected for further investigations. prepared vesicles. The persistent slower release phase is due to RSV progressively diffusing through the bilayers and into the media [66, 67]. The linear regression analysis of the mathematical mod- In Vitro Release Study els used for RSV release data from the selected vesicular formulations revealed that correlation coefficient (R ) val- Drug release from a delivery system is a standard quality control test for verifying consistency of the final product in ues of all formulations were best fitted to Higuchi’s model. The Peppas equation was used for further understand of the order to achieve an ideal system with desired release char- acteristics [60]. Furthermore, vesicular formulations are fre- mechanism of RSV release [40, 68]. Values of the release exponent “n” for F3, F5, F8, and F10 were found to be quently subjected to in-vitro release experiments in order to anticipate their in vivo performance [61, 62]. 0.1734, 0.210, 0.195, and 0.2013 respectively (Table  2), indicating a diffusion-controlled release mechanism, i.e., Figure 1 demonstrates the release patterns of the inves- tigated nanospanlastics/niosomal formulations. It was Fickian mechanism. This comes in accordance with previous studies [69–71] where Fickian mechanism was the estimated observed that the drug release was biphasic, starting with a relatively rapid drug release that lasted for 8 h, with the release mechanism from different vesicular systems. Having optimum drug release and suitable EE%, PS, and ZP and the release of more than 70% of the entrapped RSV in nioso- mal formulations (F5 and F10). Fast release was followed nanospanlastics F8 and its conventional niosomes F10 were selected for further investigations. by a steady phase with a reduced and slow-release rate 1 3 Drug Released (%) Journal of Pharmaceutical Innovation Table 2 The calculated Code Q ± SD (%) Zero order First order Higuchi Peppas 8h correlation coefficients and 2 2 kinetics parameters of RSV R R n release profile from different F3 47.32 ± 2.87 0.5063 0.4345 0.68001 0.8767 0.1734 formulations F5 69.30 ± 2.85 0.5135 0.4595 0.68608 0.9728 0.2100 F8 63.48 ± 6.05 0.3725 0.3131 0.538155 0.8392 0.1959 F10 71.92 ± 2.45 0.3953 0.3319 0.565167 0.8806 0.2013 Free RSV 85.55 ± 7.68 0.5920 0.4699 0.7571 0.9057 0.1950 Q percent total RSV released after 8 h 8h nanospanlastics formulations. Figure 3 demonstrates elec- Fourier Transform Infrared (FT‑IR) Spectroscopy tron micrographs of different RSV-loaded nanospanlastics Analysis and niosomal formulations where the formation of nano- spherical vesicles were confirmed by morphological exami- FT-IR spectroscopy is a useful tool for finding the princi- nation of the dispersions. pal peaks of a material’s unique functional groups and their Vesicles appeared as homogenous, unilamellar, well iden- changes within a specified finger printing region [72]. The tified, and almost spherical in shape having definite smooth interaction of RSV with different components of nanospan- vesicle surface enclosing an internal aqueous core. The devel- lastics and niosomes was studied using FT-IR. The FT-IR opment of closed bilayer vesicles in water may be owing to the spectra of Span 40, RSV, and selected RSV-loaded nano- amphoteric character of nonionic surfactants (Spans), which spanlastics and niosomal formulae are shown in Fig. 2. The causes the hydrophobic part to be oriented away from the aque- pursue spectra were achieved at a wavenumber ranging −1 ous environment while the hydrophilic part kept in touch with between 4,000 and 650  cm [73]. In brief, Span 40 spectrum showed characteristic peaks of −1 −1 −1 −1 2920  cm, 2921  cm, 2926  cm , and 2929  cm , which 01000 20003000 4000 indicate the carboxylic acid functional group. RSV spectrum − 1 showed a peak at 3293  cm assigned to the vibrations of its free 0.996 -O–H stretching. It also showed three typical strong absorption 0.913 F10 −1 −1 −1 bands at 1605 cm ,1583  cm , and 1380 cm corresponding 0.830 to C–C aromatic double bond stretching, C–C olefinic stretch- ing, and C–C stretching, respectively (benzene skeleton vibra- 0.747 tions) [74, 75]. The other peaks obtained at 674, 828, 966, 1147, 1.02 −1 and 1588  cm were attributed to –O–H, –C– –C–H, –C–O, 0.85 –C–C–, and C– –C bonds of benzene ring, respectively [76]. F8 0.68 The IR spectrum of the optimized RSV-loaded nanospan- lastics and niosomes (F8 and F10) showed a minor shifting 0.51 and decreased intensity in the characteristic’s peaks of both 1.04 RSV and different components. The minor changes in the 0.91 characteristic peaks of RSV might be attributed to the occur- rence of physical interaction between RSV and non-ionic 0.78 surfactant, such as Van der Wall bonds, hydrogen bond, or 0.65 RSV dipole interactions, with no chemical changes in RSV struc- 0.52 ture after encapsulation which can lead to optimum entrap- 0.96 ment of RSV within nanospanlastics [77]. It is reasonable to believe that RSV’s molecular structure has not changed 0.72 and that it will act normally in encapsulated forms. RSV had 0.48 Span 40 been successfully encapsulated, as evidenced by the small 0.24 changes in intensity [78]. 01000 20003000 4000 Transmission Electron Microscopy (TEM) -1 Wavenumber (cm ) TEM studies were carried out in order to gain a bet- Fig. 2 FT-IR spectra of free RSV, Span 40 and selected RSV-loaded ter understanding of the morphology of the investigated nanospanlastics and niosomal formulae (F8 and F10) 1 3 Transmittance Journal of Pharmaceutical Innovation Fig. 3 TEM of RSV-loaded nanospanlastics and niosomal formulae: a F8 and b F10 the aqueous environment [79]. To reduce their surface free formulations, oral administration of RSV-loaded nanospanlas- energy, vesicles must have a tendency to form spherical shapes tics (F8) prior to LPS injection showed a significant decline [80]. The nonaggregated structure of the vesicles could also in the serum levels of the ALT, AST, and creatinine by 22%, be linked to the high repulsive interactions between negatively 18%, and 24%, respectively. On the other side, pretreatment charged surfaces confirmed by ZP results. with F10 significantly suppressed the ALT serum level by 21%, yet it did not induce any significant alteration in the AST and creatinine levels, as compared to the LPS group (p < 0.05). In Vivo Studies Interestingly, the efficacy of F8 was similar to a great extent to that of the dexamethasone treated group. Sepsis-induced multiple organ damage is considered one of the major reasons for morbidity and mortality worldwide. Assessment of Oxidative Stress and Inflammatory LPS is a potent cytotoxic inducer of inflammation, which Markers lead to pathological syndrome known as endotoxic shock in animals that mimic the septic shock syndrome in humans It has been reported that LPS activates a cascade of signal- [81]. Attempts have been done to counteract the deleterious ing pathways that stimulate the release of proinflammatory effects associated with sepsis; hence, the current study was cytokines which in turn activate migration of neutrophils, constructed in employing the RSV-loaded nanospanlastics macrophages, and dendritic cells. The infiltrated immune via oral route, as a therapeutic strategy in LPS-induced endotoxemia model. Table 3 Effect of RSV formulations on the serum levels of ALT, AST, and creatinine, in LPS-induced endotoxicity in mice Assessment of Liver and Kidney Functions Groups Parameters In agreement with previous studies, the data shown in Table 3 ALT (u/ml) AST (u/ml) Creatinine (mg/ revealed that injection of LPS led to a state of liver injury, as dl) shown by the elevated serum transaminases (ALT and AST) Normal 61.14 ± 2.688 123.60 ± 4.805 0.61 ± 0.044 activities by 60%, indicating loss of functional integrity and LPS 98.26*± 4.151 198.50*± 6.440 1.10*± 0.058 leakage of enzymes typically found in the cytosol into the Free RSV/LPS 83.16 ± 4.147 186.70 ± 4.892 0.91 ± 0.072 bloodstream [82]. It was also observed that LPS injection led # # # F8/LPS 77.11 ± 5.855 161.90 ± 4.832 0.84 ± 0.045 to a significant increase in the serum creatinine by 80%, as F10/LPS 77.61 ± 2.855 186.60 ± 6.873 1.02 ± 0.055 compared to the normal group (p < 0.05), which reflected the # # # DEX/LPS 73.14 ± 4.742 147.30 ± 5.022 0.70 ± 0.063 occurrence of kidney damage accompanied with the loss of Results are expressed as mean ± S.E. (n = 6) renal function [83]. Notably, oral administration of free RSV did not show any improvement in the estimated liver and kid- *Significantly different (p < 0.05) compared to the normal group ney functions, as compared to the LPS group. As for the RSV Significantly different (p < 0.05) compared to the LPS group 1 3 Journal of Pharmaceutical Innovation Fig. 4 Effect of RSV formula- tions on the liver and kidney contents of a MDA, b NO, and c IL-1β, in LPS-induced endo- toxicity in mice. Free RSV (50 mg/kg) and formulations (F8 & F10) were orally administered for 3 consecutive days. LPS (1 mg/kg) was injected intraperi- toneally after 1 hour from the last treatment dosage. Results are expressed as the mean ± S.E (n = 6). *Significantly differ - ent (p < 0.05) compared to the normal group; significantly different (p < 0.05) compared to the LPS group; significantly different (p < 0.05) compared to the RSV group 1 3 Journal of Pharmaceutical Innovation cells produce ROS and RNS that led to peroxidation of the assessed renal biomarkers, as compared to the free RSV membrane lipids and subsequently induce a condition of group (p < 0.05) (Fig. 4a–c). Notably, the efficacy of nano- oxidative stress which destroy the surrounding tissues and spanlastics formula (F8) was highly comparable to that of may contribute to high mortality rates [2]. Hence, the con- dexamethasone in the previously estimated liver and kidney centrations of MDA (lipid peroxidation product) and NO biomarkers (p < 0.05) (Fig. 4a–c). were estimated in the current study as indicators for the oxidative stress associated with endotoxemia. Among the Histopathological Examinations released cytokine storm, IL-1β concentration was assessed as a major proinflammatory cytokine involved in the inflam- As shown in Fig.  5, hepatic lobules of the normal group matory response induced by LPS challenge [4, 45]. Moreo- had normal architecture, consisting of radiating plates or ver, IL-1β has been shown to exert a prominent role in the strands of polygonal cells with conspicuous round nuclei. NO synthesis via activation of inducible nitric oxide syn- Sinusoids are lined with a finely arranged layer of Kupffer thase [84]. Collectively, such cascade was validated in the cells with a discontinuous layer of fenestrated endothelial current study through the elevated concentrations of MDA, cells (Fig. 5a). On the other side, hepatic lobules of the LPS NO, and IL-1β, in the liver and kidney tissues of the LPS- group showed hepatic cord disintegration, which appeared challenged group (Fig. 4a–c). as empty vacuoles aligned by necrotic hepatocytes strands. As shown in Fig. 4a–c, oral administration of free RSV In addition, mononuclear cell infiltration around the necrotic did not alter significantly the liver and kidney contents tissues mainly lymphocytes and macrophages with hyperpla- of MDA, NO, and IL-1β, as compared to the LPS group. sia of Kupffer cells was seen (Fig. 5 b) that was in line with Indeed, the lack of RSV effect could be attributed to a variety the previously reported study of Liu et al. [44]. Oral admin- of factors; LPS injection decreased the absorption of RSV istration of free RSV failed to interfere with such negative owing to the inhibition of RSV transporters [3]. Moreover, consequences, showing moderate to severe degenerative the protective effect of RSV in LPS-induced endotoxemia changes that appeared as disorganization of hepatic cords model was demonstrated in the previous studies via intra- and necrotic alterations in hepatocytes defined by ballooning peritoneal route, where RSV avoids the intestinal metabo- degeneration and apoptosis of hepatocytes. Nuclear pykno- lism and effectively alleviates the oxidative stress through sis and granular cytoplasm of hepatocytes, hepatic sinusoid inhibition of ROS and enhancing the antioxidant enzymes constriction, and Kupffer cell hyperplasia with mononuclear [6, 85, 86]. In addition, the anti-inflammatory efficacy of cell infiltration were observed (Fig.  5c). These deleterious oral RSV administration in the LPS model was previously effects were mostly eliminated in the group treated with correlated with high dose of RSV (100 mg/kg), which is nanospanlastics formula (F8), showing mild swelling of twofold higher than that used in the current study [87]. hepatocytes with hyperplasia of Kupffer cells and decline Regarding the effect of RSV formulations on  the in the number of inflammatory cells (Fig.  5d). However, assessed  liver  parameters, oral administration of RSV- treatment with noisome formula (F10) showed moder- loaded spanlastics formula (F8) diminished the elevated ate hepatocytes degeneration but less severe than the free liver contents of MDA, NO, and IL-1β by 34%, 47%, and drug treated group. Hepatic lobules showed focal necrotic 25%, respectively, as compared to the LPS group (p < 0.05). areas with mononuclear cell infiltration mainly lymphocytes Similarly, pretreatment with conventional niosomes (F10) and macrophages (Fig. 5e). Sections of the dexamethasone significantly reduced the previously estimated parameters by treated group showed mild degeneration of hepatic lobules, 26%, 54%, and 25%, respectively, as compared to the LPS almost near to that observed in the nanospanlastic-treated group (p < 0.05). Interestingly, the F8 showed prevalence group, appeared as hepatocytes swelling, hepatic sinusoid over the F10 in the estimated MDA liver content, where a narrowing, and Kupffer cell hyperplasia (Fig.  5f). normalized effect was seen in the F8 treated group (p < 0.05) Regarding the histopathological examinations of kid- (Fig. 4a–c). ney tissues, sections of normal group showed normal In terms of estimated kidney biomarkers, pretreatment architecture of glomerular capillary tufts and Bowman’s with nanospanlastics formula (F8) suppressed the MDA, capsule. The epithelial lining and arrangement of renal NO, and IL-1β concentration by 56%, 24%, and 32%, respec- tubules were intact (Fig. 6a). On the contrary, sections of tively, as compared to the LPS group (p < 0.05), while oral LPS group exhibited severe histological changes, show- administration of conventional niosomes (F10) repressed ing shrinkage of capillary tufts with widening of Bow- the estimated parameters by 30%, 28%, and 28%, respec- man’s capsule and swelling of tubular epithelial lining tively, as compared to the LPS group (p < 0.05). Remark- with constriction and occlusion of tubular lumen. Also, ably, the nanospanlastics formula (F8) exhibits a signifi- necrosis and apoptosis of tubular epithelial cell lining cantly enhanced effect than the niosomal formula (F10) in with mononuclear cell infiltration mainly lymphocytes 1 3 Journal of Pharmaceutical Innovation Fig. 5 Photomicrographs of hepatic tissue sections stained with H&E after 1 hour from the last treatment dosage.  All photomicrographs of a normal group, b LPS group, c free RSV/LPS group, d F8/LPS were taken at magnification power (X200). The following are pre- group, e F10/LPS group, and f dexamethasone/LPS group. Free RSV sented in the tissue sections: necrotic hepatocytes (arrow), swelling of (50 mg/kg) and formulations (F8 & F10) were orally administered hepatocytes (arrow head), ballooning degeneration, and apoptosis of for 3 consecutive days. LPS (1 mg/kg) was injected intraperitoneally hepatocytes (blue arrow) and macrophages were observed (Fig.  6b); such effect degeneration was observed without significant necrosis or was found to be consistent with the findings of a previous apoptosis. The glomeruli have no significant pathological investigation [83]. Pretreatment with the free drug had no alteration (Fig. 6d). However, pretreatment with noisome impact on the developed histological alterations and sub- formula (F10) partially mitigated the induced injury, in stantial tissue deterioration was found (Fig. 6c). Further- which perivascular edema and mononuclear cell infiltra- more, treatment with nanospanlastics formula (F8) highly tion associated with tubular epithelial cell necrosis and alleviates the severity of injury, showing mild histological apoptosis was seen, but less severe than the free drug- changes, which appeared in the form of swelling of tubular treated group (Fig.  6e). Mild histological change was epithelial lining. In addition, tubular epithelial cell lining observed in the dexamethasone treated groups which was Fig. 6 Photomicrographs of kidney tissue sections stained with H&E after 1 hour from the last treatment dosage.  All photomicrographs of a normal group, b LPS group, c free RSV/LPS group, d F8/LPS were taken at magnification power (X200). The following are pre- group, e F10/LPS group, and f dexamethasone/LPS group. Free RSV sented in the tissue sections: glomeruli (GL) and renal tubules (RT), (50 mg/kg) and formulations (F8 & F10) were orally administered inflammatory cell infiltration (arrow), swelling of renal tubule (arrow for 3 consecutive days. LPS (1 mg/kg) was injected intraperitoneally head), edema (E), and shrinkage of glomeruli (G) 1 3 Journal of Pharmaceutical Innovation source, provide a link to the Creative Commons licence, and indicate quite comparable to those in the nanospanlastics treated if changes were made. The images or other third party material in this group, showing shrinkage of capillary tufts with widening article are included in the article's Creative Commons licence, unless of Bowman’s space of some glomeruli and few numbers of indicated otherwise in a credit line to the material. If material is not mononuclear cell infiltration (Fig.  6f). included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted Interestingly, results of the histopathological examina- use, you will need to obtain permission directly from the copyright tions were in harmony with the biochemical data, revealing holder. To view a copy of this licence, visit http:// creat iveco mmons. the superiority of nanospanlastics in delivering a sufficient org/ licen ses/ by/4. 0/. concentration of RSV to the circulation and thereby enhanc- ing its therapeutic efficacy. References Conclusion 1. Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet. 2005;365(9453):63–78. https:// doi. org/ 10. 1016/ s0140- 6736(04) 17667-8. In the current investigation, RSV was successfully loaded 2. Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, in nanospanlastics and niosomes prepared with Span 60 or Loppnow H, et al. 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Resveratrol attenuates subacute systemic inflammation-induced 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Pharmaceutical Innovation Springer Journals

Nanospanlastics as a Novel Approach for Improving the Oral Delivery of Resveratrol in Lipopolysaccharide-Induced Endotoxicity in Mice

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10.1007/s12247-023-09711-y
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Abstract

Purpose Resveratrol (RSV) is a natural polyphenolic compound that has numerous biological effects. Owing to its poor bioavailability, only trace concentrations of RSV could be found at the site of action. Therefore, the present study was aimed at developing RSV-loaded nanospanlastics to improve its oral delivery and therapeutic activity. Methods RSV-loaded nanospanlastics were prepared using the thin film hydration technique. The developed formulations were characterized via vesicular size (VS), polydispersity index (PDI), zeta potential (ZP) measurements, fourier transform infrared (FT-IR) spectroscopy analysis and transmission electron microscopy (TEM). In vitro release profile was carried out using dialysis bag diffusion technique. In vivo study was carried out using lipopolysaccharide (LPS)-induced endotoxicity model in mice to evaluate the formulations activity. Results The results revealed the successful development of RSV-loaded nanospanlastics which exhibited EE% ranging from 45 to 85%, particle sizes ranging from 260.5 to 794.3 nm; negatively charged zeta potential (≤ − 20 mV) and TEM revealed their spherical shape. An in vitro release study showed biphasic pattern with sustained release of drug up to 24 h. In vivo results showed the superiority of RSV-loaded nanospanlastics over conventional niosomes in attenuating serum levels of liver and kidney functions (aspartate transaminase (AST), alanine transaminase (ALT), and creatinine) in LPS-induced endotoxic mice. Furthermore, both of them suppressed the elevated oxidative stress and inflammatory markers (malondi- aldehyde (MDA), nitric oxide (NO), and interleukin-1beta (IL-1β)) estimated in the liver and kidney tissues. However, the nanospanlastics showed a prevalence effect over conventional niosomes in kidney measurements and the histopathological examinations. Conclusions These findings reveal the potential of nanospanlastics in improving the oral delivery and therapeutic efficacy of RSV. Keywords Resveratrol · Nanospanlastics · Niosomes · Lipopolysaccharide · Endotoxicity Introduction that harm the host via triggering a systemic inflammatory response with the onset of septic shock and may lead to Systemic inflammation is considered a hallmark of sepsis death [2]. Upon activation of macrophages by LPS, the that can be provoked by parasites, bacteria, mycobacteria, ina fl mmatory response is initiated by a cascade of mediators viruses, and fungus but mainly by gram-negative bacteria including cytokines, mainly interleukins and tumor necrosis [1]. Lipopolysaccharide (LPS) is an endotoxin constitu- factor-α, as well as the production of reactive oxygen spe- ent derived from the cell walls of gram-negative bacteria cies (ROS) and reactive nitrogen species (RNS), followed by alteration of hemostasis that finally led to multiple organ dysfunction [3, 4]. * Mostafa Mohamed Younis Resveratrol (RSV) is a natural polyphenolic compound most_younis@hotmail.com formed by plants as a result of abiotic stresses, like ultraviolet Pharmaceutical Technology Department, National Research (UV) light exposure or existence of heavy metal ions. RSV can Centre, El-Buhouth Street Dokki, Cairo, Egypt be found in grapes, nuts, and blackberries. It is considered a Drug Radiation Research Department, National Centre novel nutraceutical, providing consumers with a wide range of for Radiation Research and Technology (NCRRT), Egyptian health benefits in addition to its basic nutritional value. RSV Atomic Energy Authority, Nasr City, Cairo, Egypt Vol.:(0123456789) 1 3 Journal of Pharmaceutical Innovation exhibits geometric isomerism, but trans-resveratrol (trans-3,4,5- safe deformable nanovesicles. Both hydrophilic and hydrophobic trihidroxystilbene) is the only isomer that shows biological activ- drugs can be delivered via nanospanlastics, where these drugs ities such as anticancer, anti-inflammatory, antidiabetic, cardio- are enclosed in the inside hydrophilic compartment and the outer protective, and antiaging [5]. The protective effect of RSV in the lipid layer, respectively [30]. Nanospanlastics differ from conven- LPS-induced endotoxemia model has been previously reported, tional niosomes in terms of their structural rigidity. Cholesterol as RSV was able to hinder the induced oxidative stress in the is known to increase the rigidity of the niosomal structure and brain, liver, and kidney [6–8] and to interfere with the inflam- make the vesicles less elastic [27]. However, the inclusion of an matory signaling cascades in such model [9]. Despite the fact EA in nanospanlastics formulations offers significant flexibility that RSV was therapeutically used in experimental models of because the size and zeta potential of the formulations can be inflammation, its limited bioavailability is regarded a limiting changed to meet specific requirements using simple and reliable factor. Combination therapy was one of the widely investigated procedures [31]. Moreover, they are more chemically stable than methods to overcome bioavailability issues such as combination conventional niosomes [32]. with bioenhancing agent or low-dose radiation [10–12]. Researchers have previously studied spanlastics as a vital Due to poor bioavailability of RSV, as it exhibits rapid drug delivery system via topical route [25, 33]. One of the metabolization and excretion, only minute amounts of free goals of the present study is to develop RSV-loaded nanospan- RSV (below 5 µg/ml) can be found in systemic circulation lastics and investigate spanlastics as an oral drug delivery sys- after 25 mg oral dose [13, 14]. Also, RSV is poorly water tem. Thereafter, employing the RSV loaded nanospanlastics soluble with a log p value of 3.1, and according to the Euro- therapeutically via oral route in LPS-induced endotoxemia pean Pharmacopeia, it is considered “practically insoluble in model, aiming to improve its anti-inflammatory efficacy. water”[15]. In addition, RSV has a short half-life of about 8–14 min [16]. Hence, there is limitation in the clinical appli- cation of RSV, which suggests the development of novel for- Materials and Methods mulations to protect and stabilize RSV from being degraded, improve its water solubility, provide a prolonged release, Materials enhance its bioavailability, and target it to definite sites [17]. It is vital to overcome RSV’s poor water solubility and Chemicals limited bioavailability in order to clinically translate its posi- tive effects. Efforts to increase the efficiency and safety of Trans-resveratrol (purity ≥ 98%) was purchased from Carl- therapeutic drugs have focused on integrating the medica- Roth (Karlsruhe, German). Lipopolysaccharide (LPS from tions into a nanocarrier [18, 19]. Nanosized drug delivery E. coli serotype 055:B5) was procured from Sigma-Aldrich systems have established special consideration aiming at (St. Louis, MO, USA). Sorbitan monostearate (Span 60) and reducing the adverse effects and enhancing the drug ther - Sorbitan monooleate (Span 80) were procured from Merck apy efficiency [20, 21]. To improve in-vivo delivery of (Schuchardt OHG, Germany). Sorbitan monopalmitate (Span RSV, various nanoparticle (NP)-based formulations have 40) and cholesterol were purchased from Sigma-Aldrich (St. been studied; these studies comprise drug encapsulation Louis, MO, USA). Tween 80 was obtained from Loba Chemie into conventional colloidal carriers such as liposomes [22], Co. (India). Kits employed for the measurement of aspartate loading into solid lipid NPs [23], and encapsulation into transaminase (AST), alanine transaminase (ALT), malondi- biodegradable polymeric NPs [24]. Yet, these nanosystems aldehyde (MDA), and nitric oxide (NO) were obtained from exhibit rigid nature that lacks deformability and flexibility Biodiagnostic (Cairo, Egypt). Enzyme-linked immunosorbent throughout their way across the biological membranes [25]. assay (ELISA) kit for the measurement of interleukin-1beta Thus, more investigations have been recently performed to (IL-1β) was purchased Abcam (Cambridge, MA, USA). Other improve their elasticity in order to increase their permeabil- used chemicals were of analytical grade. ity through different biological membranes [26]. Nanospanlastics are surfactant-based nanovesicular carriers Animals that were first set up by Kakkar and Kaur [27]. Nonionic sur - factants and edge activators are the main constituents of nano- Male albino Swiss mice weighing 25–30 g were sourced spanlastics [15]. They have a good compatibility with biological from the animal breeding facility of the National Centre for systems and a low toxicity owing to the inclusion of nonionic Radiation Research and Technology (NCRRT). Animals surfactants in their structure [28]. Edge activators act by enhanc- were acclimatized for at least one week before experiment ing the permeability and flexibility of the nanocarriers’ vesicu- in the animal facility of NCRRT. They were allowed to feed lar membranes through the biological membranes by squeezing on laboratory chow and water ad libitum. All animal experi- through different pores of the biological layers without disruption ments complied with the Animal Research Reporting of In- [29]. Nanospanlastics are biodegradable, nonimmunogenic, and Vivo Experiments (ARRIVE) guidelines and were carried 1 3 Journal of Pharmaceutical Innovation out in accordance  with the National Research Council’s of Korea). The pellet was then resuspended in 10 ml distilled Guide for the Care and Use of Laboratory Animals (NIH water till further investigation. Drug amount entrapped in the publications No. 8023, revised 1978). The in vivo study was developed formulation was detected by lysis of 1 ml of resus- performed according to the guidelines set by the Research pended pellet after addition of adequate quantity of ethanol Ethics Committee at the NCRRT (permit number: 57 A/ 21). and sonication for 5 min in a bath sonicator. The content of free RSV was then estimated employing a UV–Vis spec- Methods trophotometer (Shimadzu UV–Visible spectrophotometer, 2401/PC, Tokyo, Japan) at 305 nm. Drug entrapped amount Preparation of RSV‑Loaded Nanospanlastics was calculated, as entrapment efficiency percentage (EE%), in triplicate, according to the following equation [37]: Drug-loaded nanospanlastics were developed by the thin film Amount of entrapped drug hydration technique [26, 34] employing nonionic surfactants EE% = × 100 Total drug added (NIS) such as Span 60 or Span 40 along with cholesterol. Two edge activators (EA), namely, Span 80 or Tween 80, were used at NIS: EA molar ratios of 9:1 or 8:2. Absolute ethanol and Determination of Vesicular Size (VS), Polydispersity chloroform were used in the ratio (1:1 v/v) as a solvent to dis- Index (PDI), and Zeta Potential (ZP) Measurements solve the drug [29]. In brief, one of the selected NIS, with the edge activators, in definite molar ratios, together with 10 mg VS, PDI, and ZP of the prepared nanospanlastics and con- drug was dissolved in the organic mixture in a round bottom ventional niosomes were measured by dynamic light scatter- flask [35, 36]. The mixture of solvents was then evaporated ing (DLS) using Malvern Instruments (Malvern, UK; Nano under reduced pressure by a rotary evaporator (Rotavapor, ZS). Multimodal mode was employed in all measurements. Buchi-M/HB-140, Switzerland) to form a thin film on the ZP measurements were determined after injection of the wall of the flask. This lipid film was then hydrated in 10 ml diluted sample into the specified cell. The measurements phosphate buffer pH 7.4, kept at 60 °C, under rotation for 1 h were done inFig. 4a–c) triplicate. [37]. Conventional niosomes were also prepared for compari- son using the same method where the same steps took place but without adding edge activators. The composition of RSV- In Vitro Release Profiles loaded vesicular systems is shown in Table 1. Drug release from RSV suspension, as well as from the Characterization of RSV‑Loaded Nanospanlastics selected RSV-loaded nanospanlastics and RSV-loaded niosomes, was investigated by dialysis bag diffusion tech- Drug Entrapment Assessment nique employing a shaking water bath (Memmert, SV 1422, Schwabach, Germany). In a cellulose dialysis bag (dialysis The prepared RSV-loaded nanospanlastics were separated by tubing cellulose membrane, Sigma Co., USA; Molecular centrifugation at 8000 rpm at 4 °C for 45 min using cooling weight cutoff 12,000–14,000), an aliquot of the resuspended centrifuge (Union 32R, Hanil Co., Gyeonggi-do, Republic RSV-loaded nanospanlastics (equivalent to 2 mg RSV) was Table 1 Composition, entrapment efficiency and physicochemical properties of RSV-loaded nanospanlastics/niosomes Code Molar ratio (w/w %) EE VS PDI ZP (%) ± S.D (nm) ± S.D (mV) ± S.D Span 60 Span 40 Span 80 Tween 80 CHOL F1 9 – – 1 – 62.63 ± 0.36 794.3 ± 97.78 0.411 − 33.1 ± 5.71 F2 8 – – 2 – 49.4 ± 0.03 618.2 ± 73.30 0.371 − 30.5 ± 7.26 F3 9 – 1 – – 76.31 ± 0.04 716.2 ± 74.29 0.407 − 35.8 ± 3.04 F4 8 – 2 – – 68.96 ± 0.14 558.1 ± 77.54 0.237 − 38.9 ± 6.95 F5 1 – – – 1 85.03 ± 0.02 328.8 ± 58.10 0.339 − 21.3 ± 7.04 F6 – 9 – 1 – 55.42 ± 0.03 398.5 ± 70.64 0.316 − 33 ± 4.12 F7 – 8 – 2 – 45.84 ± 0.03 357.7 ± 47.29 0.304 − 35.7 ± 6.27 F8 – 9 1 – – 65.3 ± 0.04 361.6 ± 50.14 0.355 − 41.9 ± 6.11 F9 – 8 2 – – 62.79 ± 0.05 289.5 ± 71.90 0.432 − 34.3 ± 7.19 F10 – 1 – – 1 71.28 ± 0.09 260.5 ± 52.17 0.388 − 46.2 ± 3.60 Data are expressed as the mean ± S.D. (n = 3) 1 3 Journal of Pharmaceutical Innovation added. Both ends of the bags were sealed. Based on solubil- In Vivo Studies ity characteristics of resveratrol and to fulfill sink condition, they were placed in a release solution (media) containing Experimental Design 100 ml of phosphate buffer pH 7.4 and 30% ethanol to reach sink condition [38, 39]. The shaking water bath was set at a LPS-induced endotoxicity model was carried out in the in-vivo temperature of 37 ± 0.5 °C and a rotational speed of 100 rpm. studies to evaluate the anti-inflammatory efficacy of selected At different time intervals (1, 2, 3, 4, 5, 6, 8, and 24 h), sam- RSV formulations. Mice were divided into six groups (n = 6). ples were withdrawn and substituted with same volumes of In Groups I and II, mice received saline and served as negative fresh medium to maintain sink condition. Drug concentra- and positive controls, respectively. In Group III, mice received tions were then determined in the withdrawn samples spec- free RSV (50 mg/kg) suspended in saline [3, 12]. In Groups trophotometrically at 305 nm. The ratio of RSV released to IV and V, mice received RSV-loaded nanospanlastics (F8) the total amount in the dialysis bag was used to calculate the and RSV-loaded niosome (F10) formulations, respectively. cumulative percent of RSV released. All measurements were The dose of RSV in F8 and F10 formulations is equivalent carried out three times. to that of the free drug group. In Group VI, mice received In vitro release results were subjected to various math- dexamethasone (DEX; 2 mg/kg) and served as the standard ematical models such as zero-order, first-order, Higuchi, and anti-inflammatory drug group [43]. Peppas to study the release mechanism of RSV from the Treatments were orally administered daily for 3 consecutive prepared nanosystems. The linear regression analysis for days except Group VI which was injected intraperitoneally with the release data was assessed by Microsoft Excel Program. dexamethasone only once at the third day [3, 9]. After 1 hour The mechanism was determined using the regression coef- from the last treatment dosage, a single dose of LPS (1 mg/kg) 2 2 ficient (R ). R close to 1 was considered to be the best fit was injected intraperitoneally into all animals [44, 45] except model. The exponent “n” was determined for Peppa’s model for Group I, which served as the normal/negative control group. to determine the RSV release mechanism. According to The current dose of RSV was selected according to the previ- Peppa’s theory, if n ≤ 0.43, the drug is released via a Fick- ous study of El-Ghazaly et al. [12] as they postulated that RSV ian diffusion mechanism, if 0.43 < n < 0.85, the mechanism exhibits a submaximal anti-inflammatory effect at such dose is non-Fickian diffusion, if n = 0.85, the mechanism is case level in the carrageenan-induced acute inflammation model. II transport, and if n > 0.85, the mechanism is super-case II transport [40, 41]. Sampling Procedure Fourier Transform Infrared (FT‑IR) Spectroscopy Analysis At the end of experiment, mice were sacrificed by decapita- tion under urethane anesthesia, 5 hours post LPS injection. FT-IR spectroscopy analysis was performed for the selected The blood samples were collected, and serum was obtained formulations F8 and F10, along with individual compo- by cooling centrifugation at 4000 rpm at 4 °C for 10 min and nents (RSV, and Span 40) on a JASCO 6100 FT-IR spec- then divided into small aliquots that were stored at − 80 °C for trophotometer (JASCO, Tokyo, Japan). Potassium bromide eventual use in estimating liver and kidney functions. There- (KBr) was added to the sample first. The mixture was then after, livers and kidneys were dissected, washed with saline, compacted for 2 min using a hydraulic press at a pressure and homogenized in ice-cold PBS to prepare 20% homogen- of 200  kg/cm . At wavenumbers ranging from 4000 to ates which were centrifuged at 4000 rpm at 4 °C for 10 min. −1 400  cm , a KBr pellet manufactured for each sample was The supernatants were then distributed into several aliquots and scanned against a KBr blank pellet. stored at − 80 °C to be employed for biochemical tissue analy- sis. Parts of the livers and kidneys from each group were kept Transmission Electron Microscopy (TEM) in 10% formalin, to be used for histopathological evaluation. TEM was performed to determine the morphological charac- Evaluation of Liver and Kidney Functions teristics of the selected vesicles [42]. Samples were diluted ten folds with bidistilled water and mixed well prior to the exami- The liver and kidney functions (ALT, AST, and creatinine) were nation. A drop of the investigated formulation was put on a measured in serum via a colorimetric method using commer- carbon-coated copper grid. Then, it was air-dried at room tem- cially available kits according to the manufacturer’s instruc- perature for 10 min. Afterwards, phosphotungstic acid solution tions. The absorbance of each sample was measured using the (1% (w/v)) was dropped to the grid and dried. The grid was then Unicam 8625 UV/V spectrophotometer (Cambridge, UK). loaded to TEM (JEOL Co., JEM-2100, Tokyo, Japan). 1 3 Journal of Pharmaceutical Innovation 85.03 ± 0.022%. The results  reflect the effects of nonionic Evaluation of Oxidative Stress and Inflammatory Markers surfactant type (Span 60 and Span 40) and edge activators (Span 80 and Tween 80) on EE% for all prepared formula- Liver and kidney contents of MDA and NO were estimated tions. It could be concluded that nanospanlastics prepared using Span 60 showed higher EE% than those prepared by a colorimetric method using commercially available kits, and the absorbance of each sample was measured using a using Span 40 at the same molar ratio and same edge acti- vator. This may be attributed to the effect of the surfactant spectrophotometer, while the interleukin-1beta (IL-1β) con- tent was assessed by ELISA using commercially available alkyl chain length, which is directly proportional to the EE% [47]. Span 60 has a longer alkyl chain (C16) than Span 40 kit according to the manufacturer’s instructions, and ELISA plate reader Dynatec MR5000 (Guernsey, Channel Island, (C14); this resulted in a higher EE%. Moreover, HLB plays an important role in drug EE%; the higher the HLB of the UK) was used to measure the absorbance of each sample. surfactant, the lower will be the EE%. Since Span 60 HLB (4.7) is smaller than that of Span 40 (6.7), thus, higher EE% Histopathological Evaluation was revealed by Span 60 nanospanlastics [37]. The same was revealed, upon comparing EE% of the two conventional Tissue specimens of the liver and kidney were fixed in 10% niosomes prepared, where Span 60 niosomes (F5) showed neutral-buffered formalin. They were then trimmed, washed higher EE% than Span 40 niosomes (F10). and dehydrated in ascending grades of alcohol, cleared in The formulations F2 and F7 showed the least entrapment xylene, and embedded in paraffin blocks. Sections of 5 μm efficiencies, i.e., 49.4 ± 0.033 and 45.84 ± 0.039%, respec- were obtained by sledge microtome and stained with hema- tively, when compared to other prepared formulations. It toxylin and eosin (H&E) [46]. Images were captured and can be concluded that increasing the amount of Tween processed using Adobe Photoshop (version 8). Histopatho- 80 as edge activator led to formation of unstable bilayers, logical examinations were conducted in blind fashion. increased leakiness, and permeation of drugs enclosed in the vesicles. Also, inclusion of Tween 80, with high concentra- Statistical Analysis tions, reduces the viscosity of the formulation signifying less rigidity of the bilayer membrane. As the presence of unsatu- The results were presented as mean values with standard devia- ration in Tween 80’s alkyl chains increases the bending of tions (S.D.). One-way analysis of variance (ANOVA) was used the alkyl chains to a degree that can affect the tightness to compare statistical differences between groups, followed by of the developing vesicular membranes, these effects can the Tukey–Kramer multiple comparison test. GraphPad prism enhance membrane permeability and increase RSV escape 5 (Graph Pad Software Inc., San Diego, California, USA) was into the external phases [48]. On the other hand, the lack of used for statistical analysis. A p value of less than 0.05 was unsaturation in Span 80’s alkyl chains would allow for the considered significant. The figures were represented using the construction of more rigid vesicular membranes capable of Origin® software and the Microsoft Excel programs. reducing drug leakage [49]. The edge activator concentra- tion had a beneficial effect on drug EE% until a critical span: edge activator ratio of 4:1, in which lower drug EE % was obtained beyond this ratio [50]. Results and Discussion On the opposite side, the edge activator-free dispersions (conventional niosomes F5 and F10), which were prepared Preparation of RSV‑Loaded Nanospanlastics/Niosomes using Span 60 or 40 and cholesterol, showed the highest entrapment efficiency (85.03 ± 0.022 and 71.28 ± 0.093). RSV-loaded formulations were successfully prepared These observations reflect the important role of cholesterol employing thin film hydration technique and using different which resulted in an increase in the viscosity of the formu- nonionic surfactants and edge activators. Table 1 comprises lation and increased the rigidity and stability of the bilayer the components and the physicochemical properties of the membrane, preventing the leakiness and retard permeation developed RSV-loaded nanospanlastics/niosomes. of solutes enclosed in the niosomes [51]. Characterization of RSV‑Loaded Formulations Vesicular Size (VS), Polydispersity Index (PDI), and Zeta Potential (ZP) Measurements Drug Entrapment Assessment The mean VS, ZP, and PDI values of the developed dis- The EE% of all formulations is illustrated in Table  1, persions are presented in Table 1. Vesicle sizes of differ - where entrapment efficiencies ranged from 45.84 ± 0.039 to ent formulations were in the nanorange between 260.5 and 1 3 Journal of Pharmaceutical Innovation 794.3 nm. It could be depicted that VS of nanospanlastics prepared employing span 60 showed an increased size compared to those prepared using span 40. This increase is related to the increase of Span alkyl chain length, which results in an enhanced critical packing parameter and thus 80 an increase in vesicle size [52]. The results also reveal that smaller particles were formed upon increasing edge activa- 60 tor concentration. This might be attributed to the surfactant ability to reduce interfacial tension. A higher concentration of the edge activator decreased the surface tension, allowing F3 particle partition and produced smaller nanovesicles. Similar F5 results were reported previously [53, 54]. The positive influ- F8 F10 ence of the edge activators on the developed formulations Free RES can be observed by their lower aggregation tendency. The 05 10 15 20 25 PDI is used to determine the size distribution’s width. It Time (hr) was previously reported that when PDI values are < 0.5 this indicates homogeneity and narrow distribution [55, 56]. PDI Fig. 1 Release profiles of RSV from RSV-loaded nanospanlastics and values of the developed formulations ranged from 0.237 to niosomal formulae (F3, F5, F8, and F10) as well as free drug suspen- 0.432 revealing that the dispersed particles were homog- sion enously distributed. The potential stability of the colloidal system can be that was retained for 24 h. The same pattern was observed indicated by the ZP magnitude. It was reported that ZP val- ues >|20| usually indicate minor particle aggregation [57, with the nanospanlastics formulations F3 and F8, as fast release occurred in the first 8 h and about 40% to 60% of 58]. ZP values of all the prepared formulations showed highly negative values ranging between − 21.3 mv and − 46.2 the entrapped RSV was released. This was followed by a sustained and slow release that lasted for 24 h. Vesicular mv (Table 1), indicating a good stability. Negative ZP values for vesicles, prepared without adding any charge inducing sizes played a vital role in drug release, where vesicles with smaller sizes (F5 and F10) showed faster release than those agents, could be due to preferential adsorption of hydroxyl ions or adsorption of counter ions at the vesicle surface [59] having larger sizes (F3 and F8). This biphasic release pattern is consistent with previ- which are sufficiently high for electrostatic stabilization. Due to their highest EE% and suitable VS and ZP, RSV-loaded ously reported studies and seems to be a common feature of bilayer vesicles [63–65]. The initial rapid to moderate nanospanlastics F3 and F8 with molar ratio (Span 60 or 40: Span 80; 9:1) and conventional niosomes F5 and F10 of the release phase might be attributed to the diffusion of unen- trapped drug that may be adsorbed on the surface of the molar ratios Span 60 or Span 40: Cholesterol (1:1) were selected for further investigations. prepared vesicles. The persistent slower release phase is due to RSV progressively diffusing through the bilayers and into the media [66, 67]. The linear regression analysis of the mathematical mod- In Vitro Release Study els used for RSV release data from the selected vesicular formulations revealed that correlation coefficient (R ) val- Drug release from a delivery system is a standard quality control test for verifying consistency of the final product in ues of all formulations were best fitted to Higuchi’s model. The Peppas equation was used for further understand of the order to achieve an ideal system with desired release char- acteristics [60]. Furthermore, vesicular formulations are fre- mechanism of RSV release [40, 68]. Values of the release exponent “n” for F3, F5, F8, and F10 were found to be quently subjected to in-vitro release experiments in order to anticipate their in vivo performance [61, 62]. 0.1734, 0.210, 0.195, and 0.2013 respectively (Table  2), indicating a diffusion-controlled release mechanism, i.e., Figure 1 demonstrates the release patterns of the inves- tigated nanospanlastics/niosomal formulations. It was Fickian mechanism. This comes in accordance with previous studies [69–71] where Fickian mechanism was the estimated observed that the drug release was biphasic, starting with a relatively rapid drug release that lasted for 8 h, with the release mechanism from different vesicular systems. Having optimum drug release and suitable EE%, PS, and ZP and the release of more than 70% of the entrapped RSV in nioso- mal formulations (F5 and F10). Fast release was followed nanospanlastics F8 and its conventional niosomes F10 were selected for further investigations. by a steady phase with a reduced and slow-release rate 1 3 Drug Released (%) Journal of Pharmaceutical Innovation Table 2 The calculated Code Q ± SD (%) Zero order First order Higuchi Peppas 8h correlation coefficients and 2 2 kinetics parameters of RSV R R n release profile from different F3 47.32 ± 2.87 0.5063 0.4345 0.68001 0.8767 0.1734 formulations F5 69.30 ± 2.85 0.5135 0.4595 0.68608 0.9728 0.2100 F8 63.48 ± 6.05 0.3725 0.3131 0.538155 0.8392 0.1959 F10 71.92 ± 2.45 0.3953 0.3319 0.565167 0.8806 0.2013 Free RSV 85.55 ± 7.68 0.5920 0.4699 0.7571 0.9057 0.1950 Q percent total RSV released after 8 h 8h nanospanlastics formulations. Figure 3 demonstrates elec- Fourier Transform Infrared (FT‑IR) Spectroscopy tron micrographs of different RSV-loaded nanospanlastics Analysis and niosomal formulations where the formation of nano- spherical vesicles were confirmed by morphological exami- FT-IR spectroscopy is a useful tool for finding the princi- nation of the dispersions. pal peaks of a material’s unique functional groups and their Vesicles appeared as homogenous, unilamellar, well iden- changes within a specified finger printing region [72]. The tified, and almost spherical in shape having definite smooth interaction of RSV with different components of nanospan- vesicle surface enclosing an internal aqueous core. The devel- lastics and niosomes was studied using FT-IR. The FT-IR opment of closed bilayer vesicles in water may be owing to the spectra of Span 40, RSV, and selected RSV-loaded nano- amphoteric character of nonionic surfactants (Spans), which spanlastics and niosomal formulae are shown in Fig. 2. The causes the hydrophobic part to be oriented away from the aque- pursue spectra were achieved at a wavenumber ranging −1 ous environment while the hydrophilic part kept in touch with between 4,000 and 650  cm [73]. In brief, Span 40 spectrum showed characteristic peaks of −1 −1 −1 −1 2920  cm, 2921  cm, 2926  cm , and 2929  cm , which 01000 20003000 4000 indicate the carboxylic acid functional group. RSV spectrum − 1 showed a peak at 3293  cm assigned to the vibrations of its free 0.996 -O–H stretching. It also showed three typical strong absorption 0.913 F10 −1 −1 −1 bands at 1605 cm ,1583  cm , and 1380 cm corresponding 0.830 to C–C aromatic double bond stretching, C–C olefinic stretch- ing, and C–C stretching, respectively (benzene skeleton vibra- 0.747 tions) [74, 75]. The other peaks obtained at 674, 828, 966, 1147, 1.02 −1 and 1588  cm were attributed to –O–H, –C– –C–H, –C–O, 0.85 –C–C–, and C– –C bonds of benzene ring, respectively [76]. F8 0.68 The IR spectrum of the optimized RSV-loaded nanospan- lastics and niosomes (F8 and F10) showed a minor shifting 0.51 and decreased intensity in the characteristic’s peaks of both 1.04 RSV and different components. The minor changes in the 0.91 characteristic peaks of RSV might be attributed to the occur- rence of physical interaction between RSV and non-ionic 0.78 surfactant, such as Van der Wall bonds, hydrogen bond, or 0.65 RSV dipole interactions, with no chemical changes in RSV struc- 0.52 ture after encapsulation which can lead to optimum entrap- 0.96 ment of RSV within nanospanlastics [77]. It is reasonable to believe that RSV’s molecular structure has not changed 0.72 and that it will act normally in encapsulated forms. RSV had 0.48 Span 40 been successfully encapsulated, as evidenced by the small 0.24 changes in intensity [78]. 01000 20003000 4000 Transmission Electron Microscopy (TEM) -1 Wavenumber (cm ) TEM studies were carried out in order to gain a bet- Fig. 2 FT-IR spectra of free RSV, Span 40 and selected RSV-loaded ter understanding of the morphology of the investigated nanospanlastics and niosomal formulae (F8 and F10) 1 3 Transmittance Journal of Pharmaceutical Innovation Fig. 3 TEM of RSV-loaded nanospanlastics and niosomal formulae: a F8 and b F10 the aqueous environment [79]. To reduce their surface free formulations, oral administration of RSV-loaded nanospanlas- energy, vesicles must have a tendency to form spherical shapes tics (F8) prior to LPS injection showed a significant decline [80]. The nonaggregated structure of the vesicles could also in the serum levels of the ALT, AST, and creatinine by 22%, be linked to the high repulsive interactions between negatively 18%, and 24%, respectively. On the other side, pretreatment charged surfaces confirmed by ZP results. with F10 significantly suppressed the ALT serum level by 21%, yet it did not induce any significant alteration in the AST and creatinine levels, as compared to the LPS group (p < 0.05). In Vivo Studies Interestingly, the efficacy of F8 was similar to a great extent to that of the dexamethasone treated group. Sepsis-induced multiple organ damage is considered one of the major reasons for morbidity and mortality worldwide. Assessment of Oxidative Stress and Inflammatory LPS is a potent cytotoxic inducer of inflammation, which Markers lead to pathological syndrome known as endotoxic shock in animals that mimic the septic shock syndrome in humans It has been reported that LPS activates a cascade of signal- [81]. Attempts have been done to counteract the deleterious ing pathways that stimulate the release of proinflammatory effects associated with sepsis; hence, the current study was cytokines which in turn activate migration of neutrophils, constructed in employing the RSV-loaded nanospanlastics macrophages, and dendritic cells. The infiltrated immune via oral route, as a therapeutic strategy in LPS-induced endotoxemia model. Table 3 Effect of RSV formulations on the serum levels of ALT, AST, and creatinine, in LPS-induced endotoxicity in mice Assessment of Liver and Kidney Functions Groups Parameters In agreement with previous studies, the data shown in Table 3 ALT (u/ml) AST (u/ml) Creatinine (mg/ revealed that injection of LPS led to a state of liver injury, as dl) shown by the elevated serum transaminases (ALT and AST) Normal 61.14 ± 2.688 123.60 ± 4.805 0.61 ± 0.044 activities by 60%, indicating loss of functional integrity and LPS 98.26*± 4.151 198.50*± 6.440 1.10*± 0.058 leakage of enzymes typically found in the cytosol into the Free RSV/LPS 83.16 ± 4.147 186.70 ± 4.892 0.91 ± 0.072 bloodstream [82]. It was also observed that LPS injection led # # # F8/LPS 77.11 ± 5.855 161.90 ± 4.832 0.84 ± 0.045 to a significant increase in the serum creatinine by 80%, as F10/LPS 77.61 ± 2.855 186.60 ± 6.873 1.02 ± 0.055 compared to the normal group (p < 0.05), which reflected the # # # DEX/LPS 73.14 ± 4.742 147.30 ± 5.022 0.70 ± 0.063 occurrence of kidney damage accompanied with the loss of Results are expressed as mean ± S.E. (n = 6) renal function [83]. Notably, oral administration of free RSV did not show any improvement in the estimated liver and kid- *Significantly different (p < 0.05) compared to the normal group ney functions, as compared to the LPS group. As for the RSV Significantly different (p < 0.05) compared to the LPS group 1 3 Journal of Pharmaceutical Innovation Fig. 4 Effect of RSV formula- tions on the liver and kidney contents of a MDA, b NO, and c IL-1β, in LPS-induced endo- toxicity in mice. Free RSV (50 mg/kg) and formulations (F8 & F10) were orally administered for 3 consecutive days. LPS (1 mg/kg) was injected intraperi- toneally after 1 hour from the last treatment dosage. Results are expressed as the mean ± S.E (n = 6). *Significantly differ - ent (p < 0.05) compared to the normal group; significantly different (p < 0.05) compared to the LPS group; significantly different (p < 0.05) compared to the RSV group 1 3 Journal of Pharmaceutical Innovation cells produce ROS and RNS that led to peroxidation of the assessed renal biomarkers, as compared to the free RSV membrane lipids and subsequently induce a condition of group (p < 0.05) (Fig. 4a–c). Notably, the efficacy of nano- oxidative stress which destroy the surrounding tissues and spanlastics formula (F8) was highly comparable to that of may contribute to high mortality rates [2]. Hence, the con- dexamethasone in the previously estimated liver and kidney centrations of MDA (lipid peroxidation product) and NO biomarkers (p < 0.05) (Fig. 4a–c). were estimated in the current study as indicators for the oxidative stress associated with endotoxemia. Among the Histopathological Examinations released cytokine storm, IL-1β concentration was assessed as a major proinflammatory cytokine involved in the inflam- As shown in Fig.  5, hepatic lobules of the normal group matory response induced by LPS challenge [4, 45]. Moreo- had normal architecture, consisting of radiating plates or ver, IL-1β has been shown to exert a prominent role in the strands of polygonal cells with conspicuous round nuclei. NO synthesis via activation of inducible nitric oxide syn- Sinusoids are lined with a finely arranged layer of Kupffer thase [84]. Collectively, such cascade was validated in the cells with a discontinuous layer of fenestrated endothelial current study through the elevated concentrations of MDA, cells (Fig. 5a). On the other side, hepatic lobules of the LPS NO, and IL-1β, in the liver and kidney tissues of the LPS- group showed hepatic cord disintegration, which appeared challenged group (Fig. 4a–c). as empty vacuoles aligned by necrotic hepatocytes strands. As shown in Fig. 4a–c, oral administration of free RSV In addition, mononuclear cell infiltration around the necrotic did not alter significantly the liver and kidney contents tissues mainly lymphocytes and macrophages with hyperpla- of MDA, NO, and IL-1β, as compared to the LPS group. sia of Kupffer cells was seen (Fig. 5 b) that was in line with Indeed, the lack of RSV effect could be attributed to a variety the previously reported study of Liu et al. [44]. Oral admin- of factors; LPS injection decreased the absorption of RSV istration of free RSV failed to interfere with such negative owing to the inhibition of RSV transporters [3]. Moreover, consequences, showing moderate to severe degenerative the protective effect of RSV in LPS-induced endotoxemia changes that appeared as disorganization of hepatic cords model was demonstrated in the previous studies via intra- and necrotic alterations in hepatocytes defined by ballooning peritoneal route, where RSV avoids the intestinal metabo- degeneration and apoptosis of hepatocytes. Nuclear pykno- lism and effectively alleviates the oxidative stress through sis and granular cytoplasm of hepatocytes, hepatic sinusoid inhibition of ROS and enhancing the antioxidant enzymes constriction, and Kupffer cell hyperplasia with mononuclear [6, 85, 86]. In addition, the anti-inflammatory efficacy of cell infiltration were observed (Fig.  5c). These deleterious oral RSV administration in the LPS model was previously effects were mostly eliminated in the group treated with correlated with high dose of RSV (100 mg/kg), which is nanospanlastics formula (F8), showing mild swelling of twofold higher than that used in the current study [87]. hepatocytes with hyperplasia of Kupffer cells and decline Regarding the effect of RSV formulations on  the in the number of inflammatory cells (Fig.  5d). However, assessed  liver  parameters, oral administration of RSV- treatment with noisome formula (F10) showed moder- loaded spanlastics formula (F8) diminished the elevated ate hepatocytes degeneration but less severe than the free liver contents of MDA, NO, and IL-1β by 34%, 47%, and drug treated group. Hepatic lobules showed focal necrotic 25%, respectively, as compared to the LPS group (p < 0.05). areas with mononuclear cell infiltration mainly lymphocytes Similarly, pretreatment with conventional niosomes (F10) and macrophages (Fig. 5e). Sections of the dexamethasone significantly reduced the previously estimated parameters by treated group showed mild degeneration of hepatic lobules, 26%, 54%, and 25%, respectively, as compared to the LPS almost near to that observed in the nanospanlastic-treated group (p < 0.05). Interestingly, the F8 showed prevalence group, appeared as hepatocytes swelling, hepatic sinusoid over the F10 in the estimated MDA liver content, where a narrowing, and Kupffer cell hyperplasia (Fig.  5f). normalized effect was seen in the F8 treated group (p < 0.05) Regarding the histopathological examinations of kid- (Fig. 4a–c). ney tissues, sections of normal group showed normal In terms of estimated kidney biomarkers, pretreatment architecture of glomerular capillary tufts and Bowman’s with nanospanlastics formula (F8) suppressed the MDA, capsule. The epithelial lining and arrangement of renal NO, and IL-1β concentration by 56%, 24%, and 32%, respec- tubules were intact (Fig. 6a). On the contrary, sections of tively, as compared to the LPS group (p < 0.05), while oral LPS group exhibited severe histological changes, show- administration of conventional niosomes (F10) repressed ing shrinkage of capillary tufts with widening of Bow- the estimated parameters by 30%, 28%, and 28%, respec- man’s capsule and swelling of tubular epithelial lining tively, as compared to the LPS group (p < 0.05). Remark- with constriction and occlusion of tubular lumen. Also, ably, the nanospanlastics formula (F8) exhibits a signifi- necrosis and apoptosis of tubular epithelial cell lining cantly enhanced effect than the niosomal formula (F10) in with mononuclear cell infiltration mainly lymphocytes 1 3 Journal of Pharmaceutical Innovation Fig. 5 Photomicrographs of hepatic tissue sections stained with H&E after 1 hour from the last treatment dosage.  All photomicrographs of a normal group, b LPS group, c free RSV/LPS group, d F8/LPS were taken at magnification power (X200). The following are pre- group, e F10/LPS group, and f dexamethasone/LPS group. Free RSV sented in the tissue sections: necrotic hepatocytes (arrow), swelling of (50 mg/kg) and formulations (F8 & F10) were orally administered hepatocytes (arrow head), ballooning degeneration, and apoptosis of for 3 consecutive days. LPS (1 mg/kg) was injected intraperitoneally hepatocytes (blue arrow) and macrophages were observed (Fig.  6b); such effect degeneration was observed without significant necrosis or was found to be consistent with the findings of a previous apoptosis. The glomeruli have no significant pathological investigation [83]. Pretreatment with the free drug had no alteration (Fig. 6d). However, pretreatment with noisome impact on the developed histological alterations and sub- formula (F10) partially mitigated the induced injury, in stantial tissue deterioration was found (Fig. 6c). Further- which perivascular edema and mononuclear cell infiltra- more, treatment with nanospanlastics formula (F8) highly tion associated with tubular epithelial cell necrosis and alleviates the severity of injury, showing mild histological apoptosis was seen, but less severe than the free drug- changes, which appeared in the form of swelling of tubular treated group (Fig.  6e). Mild histological change was epithelial lining. In addition, tubular epithelial cell lining observed in the dexamethasone treated groups which was Fig. 6 Photomicrographs of kidney tissue sections stained with H&E after 1 hour from the last treatment dosage.  All photomicrographs of a normal group, b LPS group, c free RSV/LPS group, d F8/LPS were taken at magnification power (X200). The following are pre- group, e F10/LPS group, and f dexamethasone/LPS group. Free RSV sented in the tissue sections: glomeruli (GL) and renal tubules (RT), (50 mg/kg) and formulations (F8 & F10) were orally administered inflammatory cell infiltration (arrow), swelling of renal tubule (arrow for 3 consecutive days. LPS (1 mg/kg) was injected intraperitoneally head), edema (E), and shrinkage of glomeruli (G) 1 3 Journal of Pharmaceutical Innovation source, provide a link to the Creative Commons licence, and indicate quite comparable to those in the nanospanlastics treated if changes were made. The images or other third party material in this group, showing shrinkage of capillary tufts with widening article are included in the article's Creative Commons licence, unless of Bowman’s space of some glomeruli and few numbers of indicated otherwise in a credit line to the material. If material is not mononuclear cell infiltration (Fig.  6f). included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted Interestingly, results of the histopathological examina- use, you will need to obtain permission directly from the copyright tions were in harmony with the biochemical data, revealing holder. To view a copy of this licence, visit http:// creat iveco mmons. the superiority of nanospanlastics in delivering a sufficient org/ licen ses/ by/4. 0/. concentration of RSV to the circulation and thereby enhanc- ing its therapeutic efficacy. References Conclusion 1. Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet. 2005;365(9453):63–78. https:// doi. org/ 10. 1016/ s0140- 6736(04) 17667-8. In the current investigation, RSV was successfully loaded 2. Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, in nanospanlastics and niosomes prepared with Span 60 or Loppnow H, et al. 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Journal

Journal of Pharmaceutical Innovation – Springer Journals

Published: Sep 1, 2023

Keywords: Resveratrol; Nanospanlastics; Niosomes; Lipopolysaccharide; Endotoxicity

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