Bioinspired Dual-Functional Solid Lipid Nanoformulations for Targeted Drug Delivery and Sustained Release for Enhancement of Potency of Albendazole, an Antihelminthic Drug

Targeted delivery has not been achieved for anthelmintic treatment, resulting in the requirement of excess drugs dose leading to side effects and therapeutic resistance. Gastrointestinal helminths ingest lipid droplets from digestive fluid for energy production, development, and defense. Worm’s habit inspired us to develop biocompatible, oral administrable, bee-wax derived solid lipid nanoparticles (SLN) with excellent drug (albendazole) loading efficiency of 83.3 ± 6.5 mg/g and sustained-release properties (86.4 ± 3.9 % of drug released within 24 h). Rhodamine B-loaded SLN showed time-dependent release and distribution of dye in vivo in Haemonchous contortus. The intestinal sustained-release property was shown by the particles that caused enhancement of albendazole potency for up to 50 folds. Therefore, this formulation has immense potential as an anthelminthic drug delivery vehicle that will not only be able to reduce the dose but will also reduce the drug-induced side effects by enhancing the bioavailability of the drug. Highlights Albendazole-loaded Solid Lipid Nanoparticles (SLN-A) were formulated using Beeswax as stating material showing high drug loading capacity of 83 mg/g with sustained-release properties and 84 ± 3 % of drug release within 24 h. SLN-A particles showed 50 fold enhancement of Albendazole activity against Haemonchous contortus worm. Rhodamine B-loaded SLN particles showed the specific uptake and in-vivo sustained release of dye in the worm.


INTRODUCTION
Of all the infectious pathological diseases present in humans and livestock, helminthic infection is one of the most common types. 20% of the world's human population, almost all wild animals, and a significant portion of the farm animal in developing countries are infected with intestinal helminths. Even though the mortality rates in these infections are less, it has been shown to cause various health complications like diarrhea, malabsorption, blood loss, and reduced growth rates (Awasthi et al., 2003; "WHO | Prevention and control of intestinal parasitic infections: WHO Technical Report Series N° 749," 2016). In some cases, they can cause anemia and retarded growth in children, adversely affecting cognition and educational abilities. In addition, the economic loss due to these soil-transmitted nematode infections results in a reduction of milk, dairy products, wool, meat, etc. (Flach, 2008;Weber, 2015). Of these intestinal worms, Haemonchus contortus is a type of blood-feeding nematode belonging to the Trichostrongyloidea family. It is present in small ruminants and causes haemonchosis and severe damage in the mucous membrane of the abomasum, resulting in gastric hemorrhage (Gasser and Samson-Himmelstjerna, 2016;Gelberg, 2017;Sendow, 2003). Although this worm is predominant in the tropical and subtropical regions and infects sheep and goats, a few cases of human infection were reported in human populations of Australia and Brazil (Sendow, 2003). H. contortus is known to absorb lipids from the GI tract of the host that is used for energy production, synthesizing parasite-specific glycerolipids and phospholipids, which are involved in host-parasitic interactions (Wang et al., 2020). In addition, a large amount of triacylglycerol was found in parasitic eggs which are used as a direct energy supply for egg development and are actively accumulated in the form of fats in the embryos (Wang et al., 2020). The uptake of lipids by the H. contortus worm from the host inspired us to make drug-loaded lipid nanoformulation for targeted delivery that will help in reducing the drug dosage and their side effects. Due to the presence of lipid core, the worms will specifically uptake the particles, taking the drug alongside and thereby increasing its potency.
A limited number of drugs, belonging to the macrolides and benzimidazoles family, are known to act against these intestinal worms. Albendazole (Abz) is a type of benzimidazole drug and was developed in 1975. It has been used to control a wide variety of parasitic nematodes in humans and animals for decades (Barrère et al., 2012). Due to its poor water solubility, the Abz drug is usually administered along with a fatty meal (García et al., 2014). The mechanism of action involves its binding to β -tubulin at the colchicine's-binding site, causing inhibition of the polymerization leading to the parasite's death (Jordan and Wilson, 2004;Kumar et al., 2013). But in recent days, chemoresistance due to altering tubulin isotype has been reported on several occasions for human and animal parasitic worms (Barrère et al., 2012;Sallé et al., 2019). Furthermore, the recommended dose of Abz against H. contortus is 10 mg/kg and 7.5 mg/kg of body weight for humans and cattle, respectively according to FDA ("CFR -Code of Federal Regulations Title 21," 2020; Goel et al., 2020).
However, the use of such a high dosage leads to side effects like headache, nausea, vomiting, stomach pain, dizziness, fever, loss of liver function, etc. (Samuel et al., 2014).
Different types of nanocarrier systems like carbohydrate-based, protein-based, or lipid-based carriers are being used, of which lipid-based drug delivery is recent and is known to have the advantage of better encapsulation efficiency, better solubility, and low toxicity (Jannin et al., 2008). Biocompatible lipids like stearic acid, cetyl palmitate, cetyl alcohol, trimyristin, glyceryl monostearate, etc. have been used for the formation of SLNs (Dastidar et al., 2019;Dolatabadi et al., 2015;Wissing et al., 2004). In the present study, beeswax was used as a lipid source, which is a non-toxic natural wax known for its effectiveness against ringworm, jock itch, and fungal skin infections (NS, 2004). Beeswax constitutes-four unsaturated acids (oleic, linoleic, palmitoleic, and linolenic), two saturated fatty acids (tetracosanoic and palmitic), some other hydrocarbons, and wax esters. In this, free fatty acids account for 12%-14% with a chain length of C24-C32 and free fatty alcohols (1%) have C28-C35. Linear wax monoesters and hydroxymonoesters are 35%-45% with C40-C48 and are derived from palmitic, 15-hydroxypalmitic, and oleic acids. Complex wax esters are 15%-27% and contain 15-hydroxypalmitic acid or diols linked to other fatty-acid molecules through their hydroxyl groups (Fratini et al., 2016).
Ethanol extract of beeswax was prepared using soxhlet apparatus that helped in the extraction of all the free fatty acids (Buchwald et al., 2009). This was later used for Abz-loaded Solid Lipid Nanoparticles (SLN-A) formulation tested against the intestinal parasitic worm H.
contortus. These particles are water-soluble, showed sustained drug release, are worm targeted, show increased bioavailability of the drug inside the parasite, and thereby enhance the drug potency. Abz-loaded SLNs were made biocompatible using Poloxamer 407 coating on them. Further, the effectiveness of the formulation was compared with the FDA-approved oral Abz solution for its activity. To confirm the uptake of drug-loaded SLN particles, Rhodamine B-loaded nanoparticles were prepared and observed for the route of ingestion and diffusion of the drug inside the worm body.

Synthesis of SLN, SLN-A, and SLN-Rh particles
Abz-loaded solid lipid nanoparticles (SLN-A) were fabricated using the double emulsion technique. The ethanolic extract of beeswax was made using the soxhlet apparatus at 78 o C. These extracts were then dried overnight in a hot air oven at 37 o C. The dried extract was later dissolved in chloroform (50 mg/ml) to make a lipid emulsion. Abz (100 mg/ml) was mixed in this lipid emulsion, which was later mixed in a 2% aqueous Poloxamer 407 solution (w/v). Finally, the solvent was Rota-evaporated to get SLN-A particles in powder form ( Fig.   1).
For the preparation of SLN particles (without drug), the particles were prepared using the above procedure without loading any drug. In addition to that, Rhodamine B-loaded SLNs (SLN-Rh) particles were also prepared using the same method. Rhodamine B (20 mg/ml) was mixed in the lipid emulsion for the formation of particles.

Particle size and Morphological Analysis
The hydrodynamic size of SLN and SLN-A were determined using Dynamic Light Dried samples were gold coated for 20 min before imaging. Fourier Transform Infrared Spectroscopy (FTIR, Agilent Technologies Cary 800 Series, Australia) was employed to check and confirm the drug-loading and formation of particles where the sample was scanned in powder form in the range 4000-800 cm -1 .

Drug Loading and Release Kinetics
Abz drug was encapsulated in SLN-A particles and its drug loading capacity was calculated and the release kinetics was observed for SLN-A and SLN-Rh particles. The particles (1 mg/ml) were taken in 30 ml PBS buffer (pH 7.4) where the system was maintained at 37 , and a spin of 130-150 rpm was applied. Samples were collected at regular intervals and checked for their absorbance at 295 nm for SLN-A particles and 555 nm for SLN-Rh particles using a UV-Visible spectrophotometer (UV-2600, UV-VIS Spectrophotometer, Shimadzu). Later, the percentage drug release and per hour percentage drug release were calculated and plotted against time and noted for its trend.

Biocompatibility of SLN /SLN-A/SLN-Rh with human cells
Albendazole and different drug/dye-loaded SLN particles (SLN, SLN-A, and SLN-Rh) were checked for their cytotoxicity using an MTT assay. Hek293 (Human Embryonic Kidney cell line) was treated with different concentrations of 5, 50, and 100 μ M and incubated for 24 h in a 5 % CO 2 incubator at 37 and humidified conditions. 10 μ l of MTT was added and incubated for 3 h. DMSO was later added to dissolve the Formazan crystals formed, and its OD was taken using an Elisa plate reader (PowerWave XS2, BioTek) at 570 nm.
The percentage of viable cells was calculated using:

Isolation of adult Haemonchus contortus worm
Adult H. contortus worms were identified and isolated from the abomasum samples of goats that were collected and brought from the slaughterhouse. These worms were first washed using a 1X PBS buffer of pH 7.4 and then later transferred to petri-plates containing RPMI media. Identification of these worms was done using the compound microscope and for further experiments, worms were selected randomly.

Treatment with SLN-Rh particles
Randomly selected 10 worms were added to RPMI media taken in 35 mm petri-plates and 20X magnification.

Treatment of worms with SLN-A particles
Worms were taken in petri-dishes containing RPMI media. For analysis of Oxidative Stress production, 6 male and 6 female worms were randomly picked and placed in a petri-dish containing RPMI media. SLN-A particles (10 µM) were added from the stock solution. The Abz drug and SLN particles were also taken as a control using the same concentration. After incubation for 3 h, the worms were washed with PBS buffer and then incubated with DCFDA dye (10 μ M) for 10 min in the dark. Later then the worms were observed under Dewinter Fluorescent Microscope at 100X and 200X magnification.

Statistical analysis
Data were presented as the mean of at least three independent experiments. Statistical analysis of data was conducted by a Student's t-test, by using MS Excel, and two measurements were statistically significant if the corresponding p-value was < 0.01.

Structure and Morphology of SLN
The SLN and drug/dye-loaded SLN particles were formed using the double emulsion technique. The flakes of SLN particles were lime-colored, orange-colored for SLN-A, and reddish for SLN-Rh particles (Fig. 1). These particles were found to be stable at room temperature in dry conditions. DLS data showed that the flakes of SLN-A and SLN were in the range 257 ± 98 nm and 165 ± 103 nm, respectively (Supplementary Information, S1. A-B). Further, the morphology of the SLN particles was confirmed using FE-SEM. The FE-SEM images showed that SLN-A particles had cuboidal-shape and their sizes range 93 ± 29 nm ( Fig. 2A-B). The images also showed that these particles were porous and had a pore size of about 3 nm (Fig. 2 C).

FT-IR study for confirming Abz loading
The FT-IR spectra were generated to determine the changes in SLN particles after the drug loading, which was clearly shown by the presence of characteristic peaks of Abz and SLN particles in SLN-A particles, proving that the Abz drug has been loaded into the particles. FTIR spectra showed a broad peak in SLN-A particles, and Abz molecule at 3313 cm -1 and a shift in the peak of SLN-A particles caused due to the N-H stretching at 3361 cm -1 from the N-H stretching at 3313 cm -1 in the Abz molecule. This peak was not observed in SLN particles. Another characteristic peak of the benzimidazole molecule was found in both particles due to the C=N stretching where Abz showed peaks at 1618 cm -1 , and SLN-A showed the peak at 1647 cm -1 (Fig. 2D-E). Another peak at 2877 cm -1 was observed in SLN and SLN-A particles due to C-H stretching. The peak for C-O stretching was observed at  Table S1).

Drug Loading and Release Kinetics
The drug loading capacity and release kinetics of SLN-A and SLN-Rh particles were studied where the drug was loaded as 83.3 ± 6.5 mg/g in SLN-A particles, and Rhodamine B was loaded as 33.3 ± 4.2 mg/g in SLN-Rh particles. The release kinetics of both Abz and Rhodamine-B was studied from SLN-A and SLN-Rh particles at the physiological pH of 6.4 and physiological temperature of 37 . Sustained drug release was observed in SLN-A particles for 24 h, and burst release followed by sustained drug release was observed for the first 12 h in SLN-Rh particles. Most of the drug was released by 48 h. SLN-A particles were suspended in a dialysis membrane with a concentration of 1 mg/ml, and the OD value was observed at 295 nm after particular time intervals (Supplementary Information, Fig. S1. C).
It was observed that 86.4 ± 3.9 % of the drug was released from SLN-A particles by the end of 48 h.
On the other hand, in the case of SLN-Rh particles, OD value was observed at 555 nm after particular time intervals, and it was noted that up to 50 % of the Rhodamine B dye was released in 2.5 h, and the almost total drug was released by 24 h (93.5 ± 2.7 %) (Fig. 3A).
This difference was observed due to the presence of polarity difference of Abz molecules and Rhodamine B molecules. Abz, due to its hydrophobic nature, is practically water-insoluble.
On the other hand, in SLN-A particles, Abz is inside the lipid layer surrounded by a poloxamer molecule with its hydrophilic tail on its outer surface. Due to this, SLN-A particles have shown sustained release of the Abz drug. On the contrary, in the case of SLN-Rh, Rhodamine B is water-soluble due to its hydrophilic nature and hence has shown burst release in the first 6 h followed by sustained release of the dye molecule (Fig. 3B).

Biocompatibility of SLN particles
The biocompatibility of these SLN particles was studied using the cell viability assay, i.e., MTT assay. Hek293 cells were treated with SLN and drug/dye-loaded SLN particles and checked for their viability. SLN, SLN-A particles, Abz drug, and SLN-Rh particles were treated at different concentrations of 5, 50, and 100 μ M (Fig. 3C). No toxicity was observed for the SLN, SLN-Rh, SLN-A particles, and Abz drug compared to control untreated cells.
Cell viability was found to be 103.0 ± 6.4 % for SLN particles, 97.8 ± 6.7 % for SLN-A particles, 98.1 ± 5.7 % for Abz alone, and 104.2 ± 3.5 % for SLN-Rh particles (Using equation (i). Thus, these particles show no toxicity towards normal cell lines, and they can be used as a potential drug delivery system towards parasitic helminths.

Treatment with SLN-Rh particles
H. contortus worms were treated with SLN-Rh and checked for their uptake and distribution of dye with respect to time in H. contortus worms. These worms were treated with 20 µM concentration for 1 h. Then images were taken in the red light filter after different time slots -1, 2, 3, and 24 h (Fig. 4). An increase in fluorescence intensity was detected in worms with increasing time showing slow drug release inside the worm. Images were also taken in a blue light filter at 3 h, which showed how the fluorescence was observed more inside the alimentary canal, which was not so clear in images taken in the red light filter due to bright intensity ( Supplementary Information, Fig. S2). Fluorescence initially observed in the alimentary canal brightened with time and was later diffused into the whole body, as seen in 24 h images. The images show that the worm first took up the particles, then the drug was released inside the alimentary canal, and later the drug was diffused in the worm body.
Treatment of worm with SLN-Rh particles showed the route of particles and dye inside the body with increasing time.

Anti-helminthic activity of SLN-A particles
Adult Motility and Morbility Assay (AMMA) was performed for testing the antihelminthic activity of SLN-A particles, where the death and paralysis time of H.
contortus worms were noted upon its treatment. 15 adult males and 15 adult females each were utilized in triplets, and it was observed that at 10 µM concentration, 66 % of the worms were paralyzed in 12 h and 100% of worms were dead within 24 h of treatment at 20 µM SLN-A particles. As a control Saline, PBS (pH 7.4) and RPMI media were used, and no deaths were observed for 24 h. In the case of Abz alone, worms had started showing paralysis at 6 h, and deaths were observed after 12 h for the 1 mM and 2 mM concentrations, and no death was observed for the lower concentrations till 24 hours. While in the case of SLN-A, lower concentrations like 5 µM showed paralysis at 6 h and death at 12 h. After 24 h, almost all the worms were found dead, showing better outcomes than in Abz alone ( Table 1). For adult male and female worms, the LD 50 values were 1 mM and 10 µM for Abz and SLN-A particles treatment.

Reactive Oxygen Species (ROS) stress generation due to SLN-A treatment
Oxidative stress generation in the adult worms due to the treatment of Abz, SLN, and SLN-A particles was checked using ROS assay (Fig. 5). After the incubation with particles, the worms were stained with DCFDA dye and the images were taken in the violet light filter and checked for ROS generation. The images were taken at the anterior, middle, and posterior end of the adult worms, and the difference in intensity of fluorescence showed the amount of stress generated. In SLN-A and Abz treated worms, high intensity of green fluorescence was observed, showing greater production of oxidative stress and more damage to the tissues. Among these, SLN-A particles showed greater ROS production with pixel intensity of 113.2 x 10 4 ± 36.3 when compared to the worms treated with Abz alone showing pixel intensity of 102 x 10 4 ± 27.1 ( Supplementary Information, Fig. S3). On the other hand, very little ROS production was observed in Control worms (RPMI media) with pixel intensity of 43 x 10 4 ± 8.9 and SLN treated adult worms pixel intensity of 52.9 x 10 4 ±10.2.

Discussion
Benzimidazole type of drugs have been used commonly as a treatment for parasitic worms, but with increasing cases of resistance against these antihelminthic drugs, there is an urgent need for the development of a new drug or a better drug delivery carriers option.
While the development of a new drug requires a lot of time and lots of resources, other drug delivery carrier systems can be formulated to increase the efficacy of already available antihelminthic drugs. H. contortus is a highly pathogenic intestinal parasitic worm that causes gastrointestinal infections in ruminants and sometimes in humans (Goel et al., 2021). These worms are known to uptake and utilize lipids from the hosts for egg and larval development, energy production, and synthesizing parasite-specific glycerolipids and phospholipids required for host-parasitic interactions (Wang et al., 2020). With increasing cases of antihelminthic drug resistance in the worms, drug-loaded SLN particles can prove to be of great benefit in oral drug delivery applications.
Here in this work, lipids that were extracted from beeswax were used for the formulations of Abz-loaded solid lipid nanoparticles (SLN-A). Beeswax, also known for its anti-allergenic and anti-swelling properties, have been used in pain-relieving, lowering cholesterol, reducing diaper rashes, and have an effect against ringworm and jock itch (NS, 2004). Therefore, beeswax, being the natural wax having various health benefits and a great source of lipids was used for these nanoparticle formulations. Poloxamer 407, because of its amphiphilic nature, acts as a surfactant and stabilizing agent and has been used as a copolymer on SLN particles which made them biocompatible and increased the availability of drugs inside the system.
Taking into consideration the above factors, SLN-A particles were made that showed enhanced antihelminthic activity of Abz against the resistant parasitic worm, H. contortus when compared with the drug alone. SLN-A particles not only increased the aqueous solubility of Albendazole, a BCS Class-II drug but also increased its effectiveness. These particles were specifically uptaken by the adult worm, which was further supported by SLN-Rh particles where a time-dependent Rhodamine B release was observed in the intestine and then was later diffused in the whole of the adult H. contortus worm. Specific uptake and intestinal sustained-release dramatically enhanced the activity of SLN-A particles in the intestine for up to 50 fold. The biocompatibility of these particles was checked for their cell viability on Hek293 cells using MTT assay and was found to be non-toxic on treatment with SLN, SLN-A, and SLN-Rh particles. Hence these particles showed no side effects, and the antihelminthic activity was due to the Abz drug alone inside the SLN-A particles.  Gryseels, 2001, 2000). Therefore, albendazole-loaded SLN formulations, which are target-specific and require much less drug dosage, can be utilized for other parasitic worms as well which will improve the effectiveness of the already existing drugs.     (F) Worm at 100X magnification showing diffused dye inside the whole body of worm after 24 h. This shows that the particles were ingested by the worm and the drug/dye was released which was later diffused inside the whole body.