Evaluation Of The Orally Administered Calcium Alginate Aerogel On The Changes Of Gut Microbiota And Hepatic And Renal Function Of Wistar Rats

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Mohammad A. A. Al-Najjar et al

Abstract

The present study evaluates the effect of calcium alginate aerogel as a potential drug carrier, on the liver and kidney functions, and on the gut microbiota of Wistar rats. The studies alginate aerogel was prepared in the form of nanoparticles using the jet cutting technique, and they were characterized in terms of specific surface areas, outer morphology, and particle size distribution. For the in vivo study, calcium alginate aerogel was administered orally, and liver and kidney functions were tested for one week and for four weeks in two distinct studies. During the short-term in vivo study, feces samples were collected for bacterial DNA extraction followed by 16S rRNA gene sequencing analyses to detect changes in gut microbiota. Results showed that the prepared alginate aerogel has an average BET-specific surface area of around 540 m2/g, with a pore volume of 7.4 cc/g, and a pore width of 30–50 nm. The in vivo study revealed that the levels of the studied kidney and liver enzymes didn't exceed the highest level of the normal range. The study of gut microbiota showed different patterns; certain groups of bacteria, such as Clostridia and Bacteriodia, increased during the aerogels regime and continued to increase after the aerogel was stopped. While other groups such as Erysipelotrichia, and Candidatus Sacha bacteria increased during aerogels treatment and then decreased again after one month. Members of the Bacilli class showed a unique trend, that is, after being the most abundant group (63%) at time 0, their relative abundance decreased dramatically until it reached < 5%; which was the case even after stopping the aerogel treatment.

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Introduction

Aerogels terminology was emerged for the first time in 1931 by Samuel Kistler, who defined them as the materials retaining their pore and network structure intact upon exchanging their pore liquid with gas [1]. After 60 years of their discovery, organic aerogels were proposed for biomedical and drug delivery applications due to their large inner surface area and open pore structure [2]. The outstanding features of this material class allow not just to increase the loading efficiency, but also to improve the bioavailability, stability, and release kinetics of the loaded molecules

Research on aerogels for oral drug administration initially focused on the use of silica aerogels because of their flexible and well-known sol-gel chemistry, as well as the possibilities of derivatization [3]. Nevertheless, the use of silica aerogels in pharmaceutical formulations designed for oral administration still poses certain concerns because of their limited biodegradability. Therefore, a further need arises to develop an aerogel carrier that is both biocompatible and biodegradable. This may be possible by using natural organic materials or biodegradable polymers, such as alginate [4] hyaluronic acid [5], chitosan, cellulose [6], and proteins [7].

Alginate (Alg), extracted from brown algae, is a linear copolymer composed of ß-1,4-D-mannuronic acid and α-L-guluronic acid monomers, which are interspersed homogenous or heterogeneous block-like patterns. Alginates have been used in the food and pharmaceutics industry because it is non-toxic, inexpensive, biodegradable, and biocompatible [8, 9]. Polyvalent cations, such as Ca2+, Ba2+, or Sr2+, can induce ionic cross-linking of Alg, transforming it from a soluble to an insoluble (gel) form.

Alg has been widely used in food products and as a pharmaceutical additive, such as a gelling agent and a tablet disintegrant [10, 11]. It has also been used in biomedical applications, including drug delivery and tissue engineering [12]. Like other edible dietary fibers, Alg and its oligomer derivatives are resistant to digestion by human endogenous enzymes. However, it can be digested significantly by human gut microbiota [13]. Despite its importance, little is known about gut microbiota which is responsible for the degradation of Alg.

The European Food Safety Authority (EFSA) has declared the safety of alginic acid and its salts, as well as their fermentation products. In vivo tests for the absorption and excretion of aqueous solutions and suspensions of alginic acid and its salts in animals showed their inability to be absorbed or metabolized by enzymes present in the gastrointestinal tract regardless of the form used during administration. However, they would be partially degraded by fermentation during their passage through the large intestine by the action of the anaerobic intestinal microbiota, causing caecal enlargement which was considered by the EFSA as an adaptive process related to the high doses tested as food additives [14]. Nevertheless, no previous in vivo study has examined the effect of calcium alginate (Ca-Alg) in the form of aerogel on the liver, kidney, or the bacterial community in the intestines.

Gut microbiota is an essential component of the human digestive system, as they help in the degradation of the ingested food, especially those for which the human body does not have the required enzymes [15, 16]. Gut microbiota can also inhibit the blooming of pathogenic microorganisms by competing with them on the nutrients’ resources, or by producing chemical substances to inhibit their growth. However, it has been shown that the degradation of the ingested digestible fibers, such as Alg, occurs mainly by fermented bacteria in the colon [17]. The products of this process are short-chain fatty acids (SCFA), which are beneficial for colon inflammation and act as anticancer. Brownlee et al. reported that the incubation of Alg and human fecal microflora demonstrated that most Alg degradation is accompanied by a change in the gut microbiota. Also, such incubation leads to the production of SCFA and gas after 24 hours [17].

Materials and methods

Materials

Alginic acid sodium salt was supplied by BASF, Germany. Calcium chloride (CaCl2) was purchased from Th. Geyer GmbH & Co. KG, Germany. Carbon dioxide (purity 99.5%) was supplied by Praxair (Germany), ethanol 99.8% was obtained from Carl Roth (Germany). All chemicals were used as received. Commercial diagnostic kits were used to measure ALP, and creatinine kits were purchased from Biostsystems S.A, Spain. The MO BIO’S PowerMax SoilDNA Isolation Kit was purchased from MO BIO Laboratories, USA. Deionized water was used throughout the study.

Methods

Preparation of aerogel particles.

The first step in the preparation of the aerogel particles was to prepare the stock solution; for that sodium alginate powder (Alg) was added to distilled water in a concentration of 1% w/w and was kept under magnetic stirring overnight at room temperature. To prepare the hydrogel particles, the approach described by Preibisch et al. [18] was followed, using Jet Cutter Type S from genia Lab, Braunschweig (Germany). In the jet-cutting process, the alginate solution was ejected through a nozzle (350 μm diameter) by compressed air (1–3 bar) to form a jet (mass flow rate of 1 g/s). The jet was cut in separate particles with a rotating disc (40 wires with a 100 μm of wire diameter of cutting discs, cylinder ratio of 5). After passing through the disc, the polymer solution fell downwards into the gelation bath with a CaCl2 solution of 5 g/L. To avoid agglomeration of the gel particles, the volume of the gelation bath was at least four times the total volume of the processed biopolymer solution, and the content of the baths was stirred with a magnetic bar (60 rpm). The separation distance between the nozzle and the gelation bath was kept at 50–70 cm. After collecting the gel particles, the stirring was continued for 60 min to ensure complete gelation and to avoid agglomeration. Gelled particles were then separated from the gelation bath via filtering and were proceeded for the solvent exchange.

Solvent exchange (water to ethanol) was performed on the collected particles stepwise (30,60, 90, 100% v/v ethanol). To ensure complete exchange, the density of the soaking liquid after the last solvent exchange step was measured using a density meter (DMA4500, Anton PaarCompany, Austria). The density was then recalculated into ethanol content. The resulting alcogel particles were eventually packed into a filter paper, and dried with supercritical CO2 in an autoclave at a constant temperature of 50˚C and a pressure of 120 bar. Continuous flow ofCO2 (20–80 g/min) was set until complete extraction of ethanol was done (2–4 hours). Afterward, slow depressurization of the autoclave (1–3 bar/min) was performed. Once the ambient pressure was reached, the autoclave was opened and aerogel microspheres of calcium alginate(denoted as Ca-Alg hereafter) were collected and stored in well-sealed containers under dry conditions.

Characterization of aerogel particles

specific surface areas of aerogel particles were measured via low-temperature nitrogen adsorption/desorption (BET) (Quantachrome Nova 3000e, Odelzhausen, Germany and Micromeritics TriStar II 3020, Germany). Prior to measurements, samples were dried for 20 under vacuum (< 1 mPa) at 60˚C.

Investigation of the outer morphology of the prepared aerogel particles was done via a scanning electron microscope (SEM) analysis (Zeiss Supra VP55, Jena, Germany) at an acceleration voltage of 3 kV and a working distance of 9.0–9.4 mm. Samples were gold-sputtered (10nm thickness) prior to imaging in order to minimize charging and improve the image contrast.

Particle size distribution measurements were performed three times for each sample consisting of approximately 10,000 particles. Dynamic image analysis was performed using aCamsizer XT with X-Jet module (Retsch GmbH, Germany) with air pressurized at 50 kPa.

Hepatic and renal function, and gut microbiota studies

A pilot study was done on a small number of animals divided into different groups to cover four dose levels of treatment substances. It was conducted for a short duration of time (1 week) in order to sight the maximum dose possible of Ca-Alg aerogels that has no major effects on renal or hepatic functions, and that does not demonstrate signs of morbidity or mortality. Doses were thoughtfully selected considering the reported oral LD50 of alginate (more than 5,000 mg/kg body weight) [19]. Yet the maximum feasible dose reached with minimal animal agitation was 2,000mg/Kg body weight.

In the second part of the study, the animals were exposed to a fixed-dose level for two weeks. Regarding age and weight variations and husbandry conditions during this study, the Organization for Economic Cooperation and Development (OECD) guidelines were followed (code No. 206). All experimental protocols were approved by the Ethical Committee (IRB, Approval number: 2019-PHA-12, attached in S1 File) of Applied Science Private University (ASU), Jordan. All staff members in the animal house at the ASU are well trained to monitor any changes that might occur to the animal as a consequence of the intervention. Specifically, they monitor parameters such as weight changes, abnormal behaviors, ruffled fur, reduced mobility, body posture, or expression of specific body fluid markers. In case of any serious abnormal changes in the animal status, they are allowed to use the humane end-point protocol that is approved by the Ethical committee at ASU. At the end of the experiments, rats were euthanized by exsanguination under general anesthesia using 2.5% thiopental sodium (Abbott Laboratories, North Point, Hong Kong) at 20 mg/kg intravenously.

Animal

In both short-term and long-term in vivo studies, ten-week-old, healthy Wistar Rats with an average weight of 240 ± 37 g were housed at a temperature of 21–23 ˚C and humidity of 35%–70% in controlled rooms, with 12 h light– 12 h dark cycles. Rats were identified and placed individually in clear-sided cages for ease of observation without disturbing their behavior. Wood shavings were used as bedding. Rats were fed a commercially available diet (Local Supplier, Jordan) and freshwater was freely offered.

The number of rats per group in the different experiments was selected according to the previously published work (examples; [20–22]), and depending on the strict regulations about “animal care and use” nationally and internationally, which stress on minimizing the number of animals used in research studies. These include but are not limited to, “Animal Care and Use Program” at the University of California, Berkeley (https://acuc.berkeley.edu/about.html), and the Norwegian guideline for animal use (https://www.forskningsetikk.no/en/guidelines/ science-and-technology/ethical-guidelines-for-the-use-of-animals-in-research/).

Pilot in vivo study (7 days repeated doses)

A repeated oral dose for a one-week duration was performed to define the maximum possible dose of aerogels demonstrating safety in Wistar rats. Ca-Alg aerogel was administered orally at four dose levels of 50, 100, 250, and 500 mg in corresponding to an average dose of 200, 400,1000, and 2000 mg/Kg (weight body weight), respectively. The results were compared with two positive control groups that were given 50 and 500 mg of pristine sodium alginate (Na-Alg)corresponding to the average dose levels of 200 and 2000 mg/Kg body weight, respectively. Eventually, all the previous groups of Ca-Alg aerogel and Na-Alg treatments were referenced to a control group (placebo). The latter group received phosphate buffer in a dose equivalent to the maximum dose when it is administered as a vehicle system for the test substances. The control and treatment groups for the pilot study consisted of 4 rats/group (two females and two males/group). The solid powders of Ca-Alg aerogel and Na-Alg were respectively suspended in phosphate buffer (pH 7.4) and mixed well directly before the oral administration because the rats refused to eat them in the dry state. For all groups, administration to rats was initiated at the same time every day using oral gavage.

Long-term in vivo exposure study (15 days repeated dose study)

Based on the results from the previous section, the dose of 250 mg of Ca-Alg aerogel was selected to be further evaluated for the long term (15 days) exposure study Wistar rats were observed for any immediate or delayed renal and/or hepatic adverse effects that might occur when exposed to Ca-Alg aerogel or when exposed to Na-Alg respectively. of. For each tested substance, three animal groups were used that included a control, treatment, and a satellite group. The control group consisted of 6 Wistar rats/group (3 males and 3 females), while the treatment and satellite groups consisted of 10 Wistar rats/group (5 males and 5 females). The Wistar rats in the treatment groups received the daily dosage of 250 mg of the test substances(Ca-Alg aerogel or Na-Alg) for 15 days, and they were sacrificed on day 16. While the satellite groups were treated for the same duration as that for the treatment groups, but were rather kept under observation for an additional 15 days post-treatment, and they were sacrificed on day 30.

It is worth mentioning that it was not possible to conduct the two studies of Ca-Alg aerogel and Na-Alg in parallel. For this reason, it was not convenient to use one control group for both. These control groups received oral phosphate buffer for 15 days only and were subsequently monitored for 14 days further. Being a short-term experiment, the solid powders of Ca-Alg aerogel and Na-Alg were also suspended in a phosphate buffer (pH 7.4) and mixed well directly before the oral administration. For all groups, the administration to rats was initiated at the same time every day using oral gavage.

Clinical and behavioral observation

In accordance with the OECD guidelines 407, animals were monitored daily for any abnormal clinical signs or changes in the behavior for the duration of the study period, with special attention at the first four hours after the administration of the test substance. Animals in the satellite and control groups were further observed for an additional 15 days without treatment administration. The satellite group allows the detection of any late-occurring clinical signs indicative of hepatic and/or renal impact through measuring Alkaline Phosphatase (ALP) and creatinine levels.

Serum biochemistry and feces analyses

Spain. The blood samples were collected from orbital sinus veins of the Wistar rats, which were then used to split up the serum. This collected serum from the samples was then transferred into sterile Eppendorf tubes and stored in the deep freeze for further testing of biochemical parameters. Feces samples were also collected every second day to investigate the change in microbial community structure. The feces were gathered in clean sterile15 ml-screw-capped tubes and were directly kept at -20˚C until all the samples were collected. For the 15-days repeated dose study, only blood samples were collected on a weekly basis to measure the levels of Alkaline Phosphatase (ALP) and creatinine, that is on day 0, day 7, day 14, day 21, and day 30 (day 21 and day 30 applied for the control and satellite groups only).

Intestinal microbial community analysis

DNA extraction from the collected feces samples was performed using MO BIO’S PowerMaxSoil DNA Isolation Kit (MO BIO Laboratories, USA), following the manufacturer's instructions. Polymerase Chain Reaction (PCR) purification and sequencing of genomic DNA (gDNA)were then conducted using Mr. DNA Lab (Molecular Research LP, USA). The PCR amplification of the 16S rRNA gene and its subsequent sequencing was done using Illumina. The 16SrRNA gene V4 variable region PCR primers ill27F mod (AGRGTTTGATCMTGGCTCAG)/ill519R and mod (GTNTTACNGCGGCKGCTG) with a barcode on the forward primer were used in 30 cycles using the HotStarTaq Plus Master Mix Kit (Qiagen, USA). After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands.

Then the pooled and purified PCR product were used to prepare the Illumina DNA library. Sequencing was performed with MR DNA on a microbiome sequencing (MiSeq) following the manufacturer's guidelines. Sequenced data were processed using the MR DNA analysis pipeline. In brief, sequences were joined, then were depleted of barcodes, after that, sequences < 150 bpas well as sequences with ambiguous base calls were removed. Sequences were denoised and operational taxonomic units (OUTs) were generated and chimeras removed. OTUs were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using BLASTn against a curated database derived from RDP-II and NCBI. A package of different statistical analyses included in “R” software was used for hierarchical clustering (version 3.5.1, vegan Package).