pH-sensitive chitosan nanoparticles loaded with dolutegravir as milk and food admixture for paediatric anti-HIV therapy

K. Priya Dharshinia,1, Hao Fangb,c,1, Ramya Devi D.a, Jin-Xuan Yangb,c, Rong-Hua Luob, Yong-Tang Zhengb,*, Marek Brzezinski´ d, Vedha Hari B.N.a,*
a Pharmaceutical Technology Laboratory, ASK-II, Lab No: 214, SASTRA Deemed-to-be-University, Thanjavur 613401, Tamil Nadu, India
b Key Laboratory of Bioactive Peptides of Yunnan Province/Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China
c College of Traditional Chinese Medicine, Yunnan University of Chinese Medicine, Kunming 650500, China d Centre of Molecular and Macromolecular Studies in Łod´ ´z, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland

A B S T R A C T

The present study aims to develop Chitosan-based polymeric nanoparticles of anti-HIV drug Dolutegravir, to aid appropriate dose adjustment and ease of oral administration as milk and food admixture for children. The isolated Chitosan from the crab shell species Portunus Sanguinolentus has been characterized for their physicochemical properties. Nanoparticles were developed with varying ratio of drug: Chitosan and assessed for particle size (140− 548 nm), zeta potential (+26.1 mV) with a maximum of 75 % drug content. Nanoparticles exhibited improved stability and drug release in the 0.1 N HCl medium compared to pure drug. The MTT assay and the Syncytia inhibition assay in C8166 (T-lymphatic cell line) infected with HIVIIIB viral strain, which showed better therapeutic efficiency and lesser cytotoxicity compared to the pure drug. In consonance with the data obtained, the use of chitosan from a novel source for drug delivery carrier has opened exceptional prospects for delivering drugs efficiently to paediatrics

Keywords:
Biopolymer
Chitosan
Dolutegravir
Spray drying
Portunus sanguinolentus
Nanoparticle

1. Introduction

HIV infections eliciting a severe immunological complaint called the Acquired Immune Deficiency Syndrome have risen as one among the severe and stimulating global public health problems (Chopra, Ni, & Lim, 2019). According to WHO statistics in 2017, around 37.9 million people living with HIV that includes a 2.8 million paediatric population (age less than <15 years) in the world (Mahy et al., 2017). The existing medical remedy, known as Highly Active Antiretroviral Treatment (HAART) is reflected as one of the substantial improvements in the ground of HIV therapy. HAART is a lifetime requisite and any dereliction leads to a faster escalation in the viral load (Sengupta & Siliciano, 2018). The main warning aspects that govern the paediatric amenability towards the HAART therapy are the caregiver barriers (forgetfulness to give the drug) and medication characteristics (bitter taste of drugs, pill burden, and the length of the treatment) (Menditto et al., 2020). Dosage form development is one critical issue to be addressed by the researchers to improve the adherence of paediatrics towards the anti-HIV treatment.
Dolutegravir (DTG), an integrase inhibitor is available in the form of oral tablets with the brand name TIVICAY at 25 mg and 50 mg fixed doses. Dolutegravir is commonly administered once daily due to its higher half-life period (14 h). On June 12, 2020, the U.S. Food and Drug Administration approved Tivicay (Dolutegravir tablets) and Tivicay PD (Dolutegravir tablets for the suspension) to treat HIV-1 infection in pediatric patients at least four weeks old and weighing at least 3 kg (6.61 pounds), in combination with other antiretroviral treatments (FDA, 2020). Treatment of AIDS by means of the conventional form of DTG is found to have certain downsides for children, due to difficulty in swallowing and variation in the paediatric dose of 5− 10 mg/day for a child weighing below 40 kg (Ruel et al., 2017). One of the appropriate means to surmount these limitations could be the processing of the pure drug with biodegradable polymeric carriers to develop as the particulate delivery system, which could be mixed along with milk/porridge for convenient administration as food admixture to children (Kersten, Barry, & Klein, 2016). The size of these polymer carrier systems in the nanometer range permits effective permeability through biological membranes, enhanced tissue compatibility, and improved cellular uptake (Mandal, Prathipati, Belshan, & Destache, 2019), thus facilitating the efficient transfer of the therapeutic agents to the target sites in vivo (Mandal et al., 2018). Chitosan (Chn) owns the standard properties of a polymeric carrier for nanoparticles like biodegradability, non-toxicity, and biocompatibility. It holds a positive charge on its surface and possesses an absorption enhancing effect and also, the size of the carriers could be optimized depending on the desired administration way (Ahmed & Aljaeid, 2016). These characteristics of Chitosan can be utilized for preparing Chn nanoparticles loaded with the required drug. Henceforth, it is hypothesised that the Chitosan isolated from novel source could be used for drug delivery applications. Therefore, the present work was attempted to formulate Chitosan nanoparticles containing anti-HIV drug DTG using the spray drying technique that could be administered along with paediatric food intake. The selected formulations shall be evaluated for process yield, zeta potential, particle size, drug content, chemical stability, solid-state transition, in vitro drug release, kinetic studies, in vitro cytotoxicity, and anti-HIV activity.

2. Materials and methods

2.1. Materials

Dolutegravir was acquired from the MSN laboratory (Hyderabad). Methanol, potassium dihydrogen phosphate, Sodium hydroxide, Hydrochloric acid, N-acetyl glucosamine, and Acetic acid (glacial), Enzymes (Pancreatin, Pepsin, and Trypsin) were purchased from Himedia. All the components and chemicals involved in this experiment are of analytical grade.

2.1.1. Cells and viruses

Human T-lymphocytes cells C8166 were donated by AIDS Reagent Project, the UK Medical Research Council (MRC), and were initially cultured in RPMI-1640 medium with FBS (10 %), Streptomycin (100 μg /mL), and Penicillin G (100 units/mL). Laboratory adapted strain HIV- 1IIIB was contributed by the AIDS Research and Reference Reagent Program, National Institutes of Health (NIH), China.

2.1.2. Collection of crab shell for Chitosan isolation

The outer shells of the three-spot swimming crab species Portunus Sanguinolentuswere acquired from local markets, Thanjavur, Tamil Nadu and authenticated by Dr. Senthilkumar TNFU, Thanjavur. Initially, the shells were thoroughly splashed with water and subsequently cleaned with the deionized water to remove all the filth on the shells. Shells were dried out in the morning light for 10 days and made into granules by grinding it in the sterile mortar and pestle.

2.2. Methods

2.2.1. Extraction of chitosan

Chitosan was isolated from the crab shells by a 3 step process such as demineralization, deproteinization, and deacetylation. The dried and granulated crab shells were exposed to the demineralization process with 5% HCl – 1:15w/v for about 36 h at 25 ◦C. The chitin acquired after the initial process of demineralization was exposed to deproteinization and deacetylation subsequently to obtain chitosan. Deproteinization of the granules was performed by treating with NaOH (5%, 1:10w/v) at 90–95 ◦C for 6 h. The deproteinized product was cleaned and washed with water until the complete removal of NaOH and dried for 15 h in a hot chamber at the temperature of 55–60 ◦C. In the final step of the extraction process, the deproteinized granules were exposed to the deacetylation reaction by treatment with NaOH (80 %-1:20w/v) at 90–95 ◦C for about5 hours. The final product chitosan (deacetylated chitin) was thoroughly washed with tap water and dried overnight in the hot chamber at 55–60 ◦C (Kumari, Annamareddy, Abanti, & Rath, 2017).

2.2.2. Characterization of extracted Chitosan by MALDI-TOF analysis

MALDI-TOF analysis is generally used to determine the molar mass distribution of the monomer/oligomer in the polymer backbone. The MALDI-TOF analysis was carried out on an Axima-Performance MALDI- TOF spectrometer (Shimadzu Biotech), armed with Nitrogen Laser with wavelength set at337 × 10− 9m. The pulsed abstraction ion source augmented the ions to the kinetic energy of 20 keV. The data were acquired in the positive linear mode, relating the accretion of 400 scans/ spectrum. The linear model calibration analysis was performed employing polyethylene glycol (PEG) in the mass range of 8000 Da as the maximum. The precision of the standardization was approximately 15 Da. The molar mass calibration was lead depending upon the average masses. Associated with the monoisotopic masses and reliant on the precision of the calibration, mass variance of 1–2 Da may be perceived. The chitosan sample was dissolved in H2O/TFA (trifluoroacetic acid) at a concentration of about 5 mg/mL. The matrix solution was prepared using Dithranol (1, 8-dihydroxy-9, 10-dihydroanthracene-9-one-10-mg/ mL) dissolved in THF (Tetrahydrofuran) at the concentration of 10-mg/ mL. The dissolved chitosan sample and the matrix were then mixed at the ratio of 2:1 (v/v). The resultant solution (about 1 μL) was dotted on a 384-well MALDI-TOF experiment plate, trailed by the vaporization of the solvent at room temperature without any aid, and 1 μL of salt solution (KCl in water – 0.05 mol/L) was dotted on the plate. The MALDI- TOF target was then investigated to yield the spectra (Menchicchi et al., 2014).

2.2.3. Degree of Deacetylation determined using UV spectrophotometric method

N-acetyl glucosamine was dissolved in 0.001 M HCl solution to obtain the 100 μg/mL standard solution. Further, a series of 10, 20, 30, 40, 50 μg/mL working solutions were prepared by diluting the standard solution and the UV absorbance (UV1800, Shimadzu) of the samples was measured @ λmax of 199 nm, using HCl solution as blank. The extracted chitosan (20− 30 mg) was dissolved in 100 mL 0.001 M HCl solution. The concentration of the acetyl group in the extracted chitosan can be calculated from the standard calibration curve and the degree of deacetylation (DDA) was calculated using the Eq. (1) (Yan, Wan, & Chai, 2019; Yuan, Chesnutt, Haggard, & Bumgardner, 2011; Sweidan et al., 2016).

2.2.4. Solid-state 13C CP/MAS NMR

The structural investigations of novel source Chitosan were executed by Solid-state Cross-Polarization Magic Angle Spinning Carbon-13 Nuclear Magnetic Resonance (CP/MAS -NMR) using Bruker-Avance AV 400 spectrometer (Bruker, Switzerland) armed with a MAS probe of 4 mm dual-resonance broad-band. About 500 mg chitosan was positioned into the spinner and introduced into the center of the magic field. The NMR spectra along with CP and MAS were documented at the frequency of 8 kHz at 295 K. A whole of over 1000 scans were accrued for a single spectrum at a recycle interval of 2 s. The degree of acetylation (DA) of chitosan was evaluated from the integral peak value of the CH3 carbon divided by the abstract integral peak value of C atom from the glucopyranosyl ring (C1–C6 atoms) (Eq. 2). The DDA was calculated using the Eq. (3) (Song, Yu, Zhang, Yang, & Zhang, 2013).

2.2.5. Comparative IR analysis of extracted chitosan and commercial chitosan

The extracted chitosan from the novel source was analyzed by Infrared spectroscopy using Attenuated Total Reflectance (ATR) mode to study its structural similarity compared to commercially available chitosan samples (Chitosan with 75 % DDA (catalog number: C3646) and the low molecular weight (LMW) chitosan (catalog number: 448869) procured from Sigma Aldrich). The analysis was performed with a Nicolet 6700 spectrometer outfitted with a deuterated triglycine sulfate (DGTS) detector. ATR was utilized for recording the spectra by including 64 sweeps at 2 cm− 1 resolution.

2.2.6. Thermal behaviour of extracted chitosan

The thermal behavior of the extracted Chitosan from a novel crab shell source was measured by employing thermogravimetry and differential scanning calorimetry (Q100, TA instrument) to discover the mass loss in the form of heat. Approximately 20–30 mg of the new chitosan sample was placed in aluminum pans and crimped using a hydraulic press. Then, the pans containing the sample were positioned on the sample-holder and the heat flow was applied from 80 ◦C up to 600 ◦C by using palladium as standard in a nitrogen atmosphere (Gohel, Sanphui, Singh, Bhat, & Prakash, 2019).

2.2.7. Preparation of chitosan nanoparticles by spray drying Chitosan-DTG nanoparticles were prepared using the Spray dryer (Version 1.0, Spraymate, Mumbai), which functions on the theory of parallel-flow nozzle spraying (the spray-dried particles and the drying air drift in the same track). The modifiable parameters comprise outlet and inlet temperature, aspirator vacuum, and the solution pump flow rate. In the present experiment, the inlet and outlet air temperatures were sustained as 140 ◦C and 55 ◦C respectively, with the pump flow rate as 11 rpm and the aspirator set at 1400 rpm (Pandey et al., 2019). To assess the effect of chitosan on drug release and solubility, the drug, and the polymer were combined in 3 ratios (Drug: Polymer – 1:1, 1:2, 1:3). The chitosan solution was prepared as 0.5 %, 1%, and 1.5 % (w/v) in 1% (v/v) glacial acetic acid, and the drug was dissolved in methanol at the concentration of 0.25 % (w/v) and the solutions were mixed before spray drying. The drug: polymer solution was then propelled into the feeding unit of the spray-dryer and the resulting powder was carried through the cyclone extractor and collected in an ampoule. The Chn nanoparticles loaded with DTG were obtained in powder form in pale white color. The sample was stored in a desiccator until further characterization (Ghanbarzadeh, Valizadeh, Yaqoubi, Asdagh, & Hamishehkar, 2018).

2.2.8. Particle size distribution and Zeta potential examination of chitosan nanoformulation

Particle size and zeta potential analysis were accomplished using the dynamic light-scattering and the charge-conductivity principle, respectively. About 10 mg of the Chitosan nanoparticles were mixed with 1 mL of distilled water and sonicated for about 10 min for the complete dispersion. The dispersion was placed in a two-way cuvette and the sample was analyzed using zeta-sizer (Malvern nano series ZS, UK) (Suganthi & Rajan, 2012).

2.2.9. FESEM: field emission scanning electron microscopy analysis of nanoparticles

The synthesized Chn nanoparticles and the pure drug DTG were analyzed through Scanning Electron Microscope with a focussed beam of electrons to compare the texture of the formed nanoparticles and pure DTG. A small amount of sample was taken for the microscopic analysis with magnification ranging from 3,000X to 100,000 × . The sample positioned on stub was sputter-coated through the thin film of gold employing auto sputter fine coater (JFC 1600, JEOL, Japan) before imaging. The sputter-coated sample was introduced into the specimen chamber and imaging was performed at an accelerating electrical energy of 3 kV (Hari, Narayanan, & Dhevedaran, 2015).

2.2.10. Swelling index of the Chitosan nanoparticles

The swelling study of the Chn nanoparticles was conducted using the procedure described by Wani et al., with required modifications. The experiment was performed on the spray-dried chitosan nanoparticles that were weighed (1 g) initially (Wb) and immersed in distilled water at room temperature (25 ◦C) for 24 h. The mixture was then centrifuged at 130 xg speed and the supernatant was discarded. The swollen weight (Ws) was obtained by weighing the pellet. Swelling index (SI) was then calculated using the following formula (Wani et al., 2020):

2.2.11. Solubility of the Chitosan nanoparticles

To confirm the solubility data and to analyze the percentage increase in the solubility of DTG after spray drying with chitosan the solubility test was performed by the procedure described by Kin et al., The free drug DTG and DC1 formulation was weighed (10 mg) and immersed in 5 mL of the following media (Distilled water, 0.1 N HCl (with and without pepsin), pH-6.8 phosphate buffer (PB) (with and without pancreatin and trypsin). The mixture was centrifuged and the supernatant was measured for the absorbance using a UV–vis spectrophotometer (UV 1800, Shimadzu) (Kim et al., 2006).

2.2.12. FTIR: fourier transform infrared spectroscopy analysis of nanoparticles

The prepared nanoparticles were subjected to FTIR analysis to study the chemical stability of the drug in presence of chitosan polymer after the spray drying process. The analysis was performed through the KBr pellet technique, in which around 5 mg of pure DTG and the drug-loaded Chn nanoparticles were individually blended with the formerly dried saturated KBr. Afterward, the pellets were shaped using a hydraulic pressure unit at 53.62 kp N cm− 2. The obtained pellets were shifted to the scanning stub and scanned at the wavenumber ranging from 4000 cm-1 to 500 cm-1(Maltesen et al., 2011).

2.2.13. XRD: X-ray diffraction analysis of nanoparticles

Any alteration in the crystalline properties of the drug in addition to chitosan polymer after the spray-drying process was analyzed using X- Ray Diffraction analysis. The spray-dried Chn nanoparticles and the pure drug were placed in the holder and exposed to the X-ray using CuKα (Rigaku, Japan), with a current (30 mA) and the voltage(40 kV). The samples were scanned in a range of 10◦ – 80◦ at 2θ (Dapiaggi, Artioli, & Petras, 2002).

2.2.14. TG-DSC: thermo-gravimetric- differential scanning calorimetric analysis of nanoparticles

The thermal behavior of the pure DTG, Chitosan, and the drug- loaded Chn nanoparticles was measured by employing thermogravimetry and differential scanning calorimetry (Q100, TA instrument). Approximately, 16–20 mg of the nanoparticles and the pure drug was individually placed in aluminum pans, which was positioned on the sample-holder and heat flow applied at 80◦C min-1 up to 900◦C, keeping palladium compound as standard in a nitrogen atmosphere (Gohel et al., 2019).

2.2.15. In vitro drug release studies of the Chitosan-Dolutegravir Nanoparticles

To confirm the amount of drug present in the spray-dried formulation, drug content analysis was performed. A stock solution of the nanoparticles was prepared with 5 μg/mL concentration and subjected to UV analysis (UV1800, Shimadzu) at 259.8 nm (Balasaheb et al., 2015). The in vitro drug release study of the DTG loaded Chn nanoparticles was compared with the pure DTG in three different media, namely distilled water (pH 7.0), phosphate buffer (PB) (pH 6.8),0.1 N HCl (pH 1.2), PB with pancreatin and trypsin, 0.1 N HCl with pepsin by dialysis bag method (Dialysis membrane Himedia, cut-off value- 12− 14 kDa). The maximum absorption of Dolutegravir was found at the proximal small intestine which is the duodenum (Cimino et al., 2018). Hence the simulated intestinal fluid (in the fed state, with Pancreatin and Trypsin from the porcine pancreas) was used as the dissolution media and the release studies were performed. Also, the simulated gastric fluid with pepsin (fast state) was tested for the release of the drug from the chitosan nanoparticles (Jantratid et al., 2008). About 10 mg of nanoformulation was mixed with 0.5 mL of the corresponding media, placed inside the dialysis bag, and immersed in 10 mL media in a vial. At every predetermined time point, the 10 mL samples were collected and immediately replaced with the fresh corresponding medium. The temperature and magnetic stirring speed of the sample were retained at 37 ◦C±2 ◦C and 200 rpm, respectively throughout the 24 h study period (Jain et al., 2008).

2.2.16. Drug release kinetics of the nanoparticles

The drug release mechanism of DTG loaded Chn nanoparticles was determined by fitting the drug release data acquired for different media to the various release kinetic simulations namely zero-order (F = k0*t), first-order (F = 100*[1-Exp(-k1*t)]), Higuchi (F = kH*t^0.5), Hixson–Crowell (F = 100*[1-(1-kHC*t)^3]), Korsmeyer–Peppas (F = kKP*t^n), Makoid Banakar (F = kMB*t^n*Exp(-k*t)), Gompertz (F = 100*Exp {-α*Exp[-β*log(t)]}), Hopfenberg (F = 100*[1-(1-kHB*t)^n]) and Baker Lonsdale (3/2*[1-(1-F/100)^(2/3)]-F/100=kBL*t). The R2 value and the n-value obtained from these models were compared to conclude the perfect fitting model for each trial. The drug release modeling was analyzed using DD solver Software (Dash, Murthy, Nath, & Chowdhury, 2010).

2.2.17. In vitro cytotoxicity assay in human cell lines

The cytotoxicity of samples on C8166 cell lines (Human T cell leukemia) was determined by means of MTT assay. About, 4 × 104 cells were plated in 96-well plates and segregated for the negative control, positive control, and six concentration of test sample for triplicate analysis. The test samples were added to the wells at predetermined serial diluted concentrations and incubated for 3 days at 37 ◦C and 5% CO2. About 20 μL MTT was further added to each well and incubated for 4− 5 hours at 37 ◦C. Then, the supernatant was discarded and further 100 μL 15 % sodium dodecyl sulfate, and 50 % dimethylformamide was added and then incubated over the night at the temperature of 37 ◦C. The absorbance value of the samples was measured using an ELx800 reader (Bio-Tek, USA) at 630 nm. Cell viability and cytotoxicity concentration (CC50) was calculated using equation 4 & 5.

2.2.18. Anti-HIV activity

The comparative efficacy of the pure drug and the drug-loaded Chn nanoparticles in terms of the inhibition of HIV cells was evaluated based on the syncytia formation assay. Viral protein facilitates the merging of an infected cell with adjacent cells leading to the development of multi- nucleate enlarged cells called syncytia. Generally, these syncytia are the upshot of expression of a viral fusion protein at the host cell membrane in the course of viral replication. The HIV inhibition effect of various concentrations of test samples was investigated. Initially, 4 × 104 C8166 cells were added with HIV-1IIIBand the cells were infected at the Multiplicity of Infection (MOI) of 0.04. The cells were cultured under infection in 96-well plates at 5% CO2and 37 ◦C for 3 days. After 3 days of infection, the cytopathic effect (CPE) was evaluated by numbering the syncytia (the multinucleated giant cells) in each well using an inverted microscope. The inhibitory effect (%) of the test samples on syncytia formation was calculated by the number of syncytia in sample treated cells related to that in HIV-IIIB infected control cells (Eq. 6). The median inhibitory concentration (IC50)value is the concentration of drug that inhibits 50 % syncytia formation, which was calculated using Eq. 7.
The selectivity index is the measure of the therapeutic effect and the safety of the given drug, eliminating the target pathogen without affecting the normal cells. For any drug formulation to be effective, the selectivity index value should be fairly higher, which means the drug kills the target first before the host (representing IC50 < CC50). The therapeutic effect of the DTG loaded Chn nanoparticle was calculated by Eq. 8 (Wang et al., 2009).

3. Results

3.1. Characterization of isolated chitosan

3.1.1. MALDI-TOF

The MALDI-TOF analysis was performed to illustrate the chemical structure of chitosan oligomers and to determine its composition consisting of glucosamine GlcN (D) and N-acetyl glucosamine GlcNAc (A) moieties. Oligomers functionalized with expected end groups (H and OH groups) and degree of polymerization (DP) ranging from 4 to 8 are observed, cationized by sodium and potassium, respectively (Fig. 1) (Trombotto, Ladaviere, Delolme, ` & Domard, 2008). The comparison of mass spectra revealed the presence of the mixture of three different oligomers: fully deacetylated oligomers (D6A0, D7A0), a mixture of acetylated and deacetylated oligomers (D1A3, D1A4, D3A1, D3A2, D3A5, D6A1), and one fully acetylated oligomer (D0A6), as shown in Fig. 1. For instance, the peak at 127 mass units could be recognized as oligomer with DP7, whereas from calculations it was found to be 1226.58 mass units (D6A1 = 6 × 161.07 (D) + 203.07 (A) + 1.00 (H) + 17 (OH) + 39.0983 (K, the cationization ion). Moreover, the fragmentation of carbohydrate structures could be detected since the sample was force degraded during the analysis to resolve the solubility issues. As a result, the loss of 18 and 59 mass units correspond to the fragmentation of C2H5ON and H2O molecules, respectively (Trombotto et al., 2008). To summarize, the chitosan isolated from Portunus Sanguinolentus consists of both glucosamine (D) and N-acetyl glucosamine (A) with the distribution of all DxAy rather in the random order, however, the population of both partially or fully deacylated species prevail. Also, based on the UV spectrophotometric method (as explained in Section 2.2.3), it was established that the DDA (%) of the extracted chitosan was about 95 %. This result is in accordance with the MALDI-TOF analysis, which again confirms the occurrence of deacetylated monomers in superior ratio than the acetylated monomers in the chitosan backbone.

3.1.2. Solid-state 13C CP/MAS NMR

The structural analysis through 13C CP/MAS NMR in the solid-state is obviously beneficial for carbohydrate polymers such as chitosan, instigating no destruction to the samples. Fig. 2A displays the spectrum of the extracted chitosan and the commercial chitosan (chitosan with 75 % DDA and the low molecular weight (LMW), Sigma Aldrich). The spectrum of the isolated chitosan is highly analogous to the commercial one with negligible deviations (Senra et al., 2017). The individual spectrum of the isolated chitosan (Portunus sanguinolentus) had displayed (Supplementary Fig. 1) defined peaks of C1 (101.47 ppm), C2 (57.07 ppm), C3 (53.26 ppm), C4 (80.92 ppm), C5 (73.52 ppm), C6 (60.87 ppm) and CH3 (20.67 ppm). The spectrum also displayed a peak at 169.86 ppm, which is assigned to the carbon atom in the C––O group. Furthermore, we have detected an extremely fragile methyl peak (20.67 ppm), indicating a relatively high DDA (Heux, Brugnerotto, Desbrieres, Versali, & Rinaudo, 2000). It is also apparent that the product is highly pure since the peaks at 174 ppm, 146 ppm, and 117 ppm, belonging to the Carbon signals of catechol and protein compounds are absent in the spectrum (Zhang, Haga, Sekiguchi, & Hirano, 2000). The DDA of the extracted chitosan and commercial chitosan was 93.2 % and 75 %, respectively. The integral area under this peak for the isolated chitosan is lesser than the commercial chitosan, which may be due to the higher DDA (%) value of the isolated chitosan. The obtained results were also in accordance with the value determined using UV Spectrophotometric method. Previous research showed that the chitosan with a high degree of deacetylation is more biologically active than chitin and low DDA chitosan (Howling et al., 2001).

3.1.3. Comparative FTIR analysis of extracted chitosan and commercial chitosan

As an influential investigation tool, FTIR analysis has been extensively adopted to detect the occurrence of definite functional groups and significant chemical bonds in a sample. The comparative FTIR spectra (Fig. 2B) of the isolated chitosan and the commercially available chitosan displayed identical peaks with mild variation in the absorption intensity values. The extracted Chitosan presented a strong band at 3354 cm− 1, which corresponds to the OH stretching vibration, the intermolecular hydrogen bonds of the polysaccharide. The absorption band at 2963 cm− 1in the commercial chitosan corresponds to the asymmetric C–H stretching vibration, which was observed with lesser intensity in the extracted chitosan. The apparent disappearance of the band at 1647 cm− 1 for the extracted chitosan revealed the successful N-deacetylation. The bands at 1374 cm− 1 were attributed to the stretching vibrations of the C–N bond. The peak at 1152 cm− 1 could be related to the OH bending vibration. The C––O stretching vibration of the chitosan was obtained at a wavenumber of 1068 cm− 1. In addition, peak at 765 and 749 cm− 1 was ascribed to the C–H out-of-plane vibration and the NH– twist vibration, respectively (Crews, Rodriquez, Jaspars, & Crews, 1998).

3.1.4. Thermal behaviour of extracted chitosan

The TG –DSC curve of the isolated chitosan showed in supplementary Fig. 1, wherein a step-wise thermal degradation of the sample was observed. An initial endothermic bend curve was perceived amid 40–100 ◦C, reaching the peak at 81 ◦C. Around 9.5 % of mass loss could be related to this consequence and it is ascribed to the evaporation of absorbed water content in the polymer. An exothermic peak at 318 ◦C could be linked to a mass loss (10 %), which corresponds to the thermal degradation of the polymeric backbone with the evaporation of volatile components. The polysaccharide is pyrolyzed and the structure starts to deteriorate by the arbitrary splitting of the glycosidic bonds (Neto et al., 2005). The second exothermic curve observed at 389 ◦C could be due to the residual degradation of chitosan. Also, the second endothermic process of the isolated chitosan occurred in between the temperature range of 300− 400 ◦C with a peak at 360 ◦C, which represented the melting point of the polymer. Finally, an endothermic peak at 752 ◦C in the DSC curve represented the completed degradation of the polymer with corresponding weight loss in the TG curve. Thermal degradation of chitosan happens via four steps; however, the exothermic process occurring at 318 ◦C is the most significant one, since it is associated with the breaking down of the polymeric backbone. Lopez, Merc´ ˆe, Alguacil, & Lopez-Delgado (2008) ´ had demonstrated that the degradation of chitosan is a multifaceted reaction, which cannot be termed in a single pair of Arrhenius parameters (Lopez et al., 2008´ ). Also, Rubini et al., 2018 had reported the XRD pattern of the chitosan extracted from Portunus Sanguinolentus that showed a unique diffraction pattern at the 2θ value of20˚, which corresponds to its crystalline property and also confirmed the structural similarity of the extracted chitosan with the commercial chitosan (CC).

3.2. Characterization of Chitosan nanoformulation

3.2.1. Particle size distribution and Zeta potential examination

The particle size and zeta potential analysis were performed for the spray-dried Chitosan nanoparticles loaded with DTG. The average particle size, zeta potential, and PDI value of the nanoparticles formulated with different ratio of drug and polymer are given in Table 1. Formulation DC1 containing 1:1 ratio of drug: polymer had shown mean particle size in the range of 140 nm–548 nm and the zeta potential value of 26.1 mV, which ensured optimum stability of the nanoformulation.

3.2.2. FESEM: field-emission scanning electron microscopy

The Scanning Electron Microscopy was performed for the selected Chn nanoparticles loaded with DTG(DC1) and compared with the pure drug (Fig. 3). The results showed the unprocessed pure DTG was rod- shaped and irregular in size, whereas the Chn nanoparticles were sphere-shaped. The particle size observed from SEM analysis could be correlated with the zeta size analysis result.

3.2.3. FTIR: fourier transform infrared spectroscopy

The FTIR analysis was performed for the pure DTG and the spray- dried Chitosan nanoparticles (Fig. 4) to confirm the presence of significant chemical bonds. The peaks for stretching bonds of O–H, C–H, and COC–– were obtained at 3434 cm− 1, 2976 cm-1, and 1258 cm− 1, respectively. The bending vibration peak of the C–H bond was observed at 856 cm− 1. The double bonds of CO and conjugated CC––– – were visualized at 1643 cm− 1 and 1539 cm− 1, respectively. The CN– bond in the pure DTG was observable at 1023 cm− 1. The OH stretch, C–H stretch, CC, COC stretch, C–H bend, CO–––––– – conjugated bond, and the C–N bonds were all also observed in the nanoparticles formulations with a very mild shift in the peak values which showed negligible chemical interactions. Therefore, the chemical nature of the drug was not affected during the spray drying process in addition to the isolated polymer chitosan. The unique peak at 1060 cm-1 for the presence of C–F bond in the pure DTG was also observed for the formulations DC1, DC2, and DC3 at 1066 cm− 1, 1061 cm-1, and 1056 cm− 1 respectively, which added significant value to the chemical stability of the polymeric nanoparticles (Osman and Arof, 2003; Albrecht et al., 2018).

3.2.4. XRD: X-ray diffraction analysis

The sharp intense XRD peaks obtained for the DTG and the isolated Chitosan confirmed the crystalline nature of the pure samples (Fig. 5). However, these peaks were absent in the nanoformulations, wherein the DC1 formulation was amorphous, whereas the DC2 and DC3 nano-particles exhibited a partial crystalline nature due to the presence of a higher ratio of the chitosan. The amorphous nature of DC1 formulation could be attributed to the solid-state transition or conversion of the crystalline materials to amorphous form during the spray drying process, which could lead to enhancement of drug solubility (Monshi, Foroughi, & Monshi, 2012).

3.2.5. Thermo-gravimetric and differential scanning calorimetric analysis (TG-DSC)

The TG-DSC analysis of the DTG pure sample and its corresponding Chn nanoparticles (Fig. 6) were compared to evaluate the thermal stability of the drug in the formed nanoparticles. The endothermic peak representing the melting peak (Tm) of pure DTG and isolated chitosan was found at 359 ◦C and 360 ◦C, respectively. However, no endothermic peaks corresponding to the melting point of the DTG and polymer were observed in DTG-Chn nanoparticles formulations. The absence of an endothermic peak in the nanoparticles could be ascribed to the solid- state conversion of pure DTG from crystalline form to amorphous form after the spray drying process in presence of chitosan. The DC1 formulation showed an endothermic peak at 80 ◦C corresponding to the evaporation of water molecules, which was absent in the DC2 and DC3 formulations due to the complete removal of moisture in the formed nanoparticles with high polymer concentration during the spray drying process(Shete, Puri, Kumar, & Bansal, 2010).

3.2.6. In-Vitro drug release study

The formulated DTG-Chn nanoparticles showed 75 ± 2%, 60 ± 3%, and 45 ± 2% of drug content for DC1, DC2, and DC3 formulations, respectively. As the drug: polymer ratio was increased from 1:1 to 1:2 and 1:3, the drug content was found to be decreasing, which could be due to the rapid drying of the polymer before encapsulation of the drug. The in vitro drug release experiment of the formed nanoparticles was carried out by the dialysis membrane method in 3 different media and compared with the pure drug. In distilled water medium, the pure drug reached 50.10 % concentration at the end of 24 h, whereas the DTG-Chn nanoformulation DC1, DC2, and DC3 had shown 36.44 %, 40.49 %, and 39.96 % of drug release, respectively (Fig. 7A). In PB media, the pure drug released 58.39 % of its total concentration, and the nanoformulation DC1, DC2, and DC3 showed 59.69 %, 54.64 %, and 66.42 % drug release, respectively (Fig. 7B). Since the nanoformulation was developed for admixture through the oral route of administration, the release profile in the acidic medium is considered as an important parameter (Ghareeb & Neamah, 2017). In 0.1 N HCl medium (stomach pH 1.2), all the formulations and the pure drug reached 100 % of drug release at the end of 24 h (Fig. 7C). Further, the nanoparticle formulations were evaluated in addition to milk (15 min), followed by the drug release studies in 0.1 N HCl media by the dialysis membrane process. The drug release pattern of all the 3 formulations (Fig. 7D) was similar, as observed in plain 0.1 N HCl media. This confirmed the stability of the formulations while added with milk and their suitability for milk admixture dosing (Alhawmdeh, Barqawi, & Alkhatib, 2018).
In order to maintain the therapeutic efficacy of the drug, a considerable amount of drug should be present in the bloodstream in vivo. To evaluate this parameter, the T30, T50, and T80 values (time taken to release 30 %, 50 %, and 80 % of its drug content) were calculated. Table II displays the time-based release of the pure DTG and the nanoformulations. The time taken to release 80 % of the drug was found to be 20 h, 6 h,9 h, and 9 h for the pure DTG, DC1, DC2, and DC3, respectively. Since the half-life of Dolutegravir is 14 h, the enhanced drug release from the nanoparticles would be suitable for once-daily therapy and improved bioavailability at a low dose in paediatric patients. The DC1 nanoparticles were considered to be optimum compared to other ratio formulations, which showed the lowest particle size and higher positive zeta potential that could probably be the reason for its faster release of the drug (Griesser et al., 2018).
The optimized formulation DC1 was further analyzed for the drug release in presence of the enzymes (pepsin, pancreatin, and trypsin). At the end of 24 h, the percentage release of pure DTG was around 47.71 ±1.0 %, 42.28 ± 2.3 %, and 44.10 ± 3.2 % in the 0.1 N HCl with pepsin, PB-6.8 with trypsin, and PB-6.8 with pancreatin respectively (Fig. 8). The release rate of DTG from the selected formulation DC1 reached a maximum of 59.49 ± 2.5 %, 62.33 ± 2.3 %, 56.82 ± 3.3 % in 0.1 N HCl with pepsin, PB-6.8 with trypsin and PB-6.8 with pancreatin respectively at the end of 24 h (Fig. 8). The presence of enzymes (trypsin and pancreatin) have not significantly influenced the release of DTG in the PB media pH 6.8 (Anal, Stevens, & Remunan-Lopez, 2006). In the case of 0.1 N HCl media (without pepsin), the nanoparticles showed 100 % drug release at the end of 24 h, whereas only 60 % release was observed in presence of pepsin. In a similar fashion, the free drug DTG showed 100 % drug release in 0.1 N HCl media (without pepsin) and 44.10 ± 3.2 % release in presence of pepsin at the end of 24 h. This could be attributed to Physico-chemical interactions between drug and pepsin (do Nascimento, Montalvao, ˜ & Aversi-Ferreira, 2012).”
The pure drug Dolutegravir is slightly soluble in water (<1 mg/mL) and methanol (0.5 mg/mL) (the solvent used for spray drying). The drug is practically insoluble in 0.1 N HCl pH 1.2 (< 0.1 mg/mL). An increase in pH to 6.8 results in a slight increase in the solubility of Dolutegravir, but remains to be very slightly soluble (0.1− 1 mg/mL) (Meergans et al., 2016). The solubility of Dolutegravir was found to be 190.18 ± 9.8 μg/mL in distilled water and 36.14 ± 5.8 % μg/mL in 0.1 N HCl media. At increasing pH of 6.8, the solubility was slightly increased to 84.04 ± 5.6 μg/mL. These results are comparable to the previously reported solubility data (Meergans et al., 2016). The presence of enzymes (pepsin, trypsin, and pancreatin) did not affect the solubility of the free drug (Supplementary Table I). After spray drying of the DTG with Chitosan, there was a great fold increase in the solubility of the drug in the various dissolution media. Although there was no increase in the solubility in distilled water (200 ± 4.3 μg/mL), the chitosan nanoparticles displayed a 20-fold increase in the solubility of the drug (400 ± 2.8 μg/mL) in the 0.1 N HCl media. This could be due to the solubility of Chitosan polymer (acid-soluble polymer) from the matrix nanoparticles, which enabled the entrapped drug to dissolve faster. The DTG-Chn nanoparticles displayed around the 3-fold increase in the solubility in the PB pH-6.8 media (262 ± 5.7 μg/mL), which is more desirable, since the drug is highly absorbed in the duodenum (pH-6.8)”

3.2.7. Drug release kinetics

Based on the values of the highest R2 and lowest SSR obtained from various mathematical models, the mechanism of drug release concerned with time was calculated. The drug release from both the pure DTG and DTG-Chn nanoparticles in three different media followed the Makoid Banakar (MB) model of kinetics. The n-value <5 represented the Fickian mechanism of drug diffusion. Since Makoid–Banakar model is a hypothetical model and not abstracted from any kinetics base, it does not sufficiently illustrate the drug release kinetic properties. Since the k- value of the Makoid–Banakar model obtained for all the media was almost equal to zero, the release kinetics equation can be fitted to Korsemeyer Peppa’s (KP) model of dissolution(Costa & Sousa Lobo, 2003). There are numerous concurrent processes considered in the KP model such as diffusion of water into the particles, swelling of the particles due to aqueous entry, the formation of gel, diffusion of the drug out of the membranes, and dissolution of the drug into the media(Costa & Lobo, 2001).
To utilize the Korsemeyer Peppa’s release kinetics parameter to determine the swelling and diffusion mechanism of the formulation in various media, the swelling index of the optimized formulation was found out. The swelling ratio of the nanoparticles was found to be 9.25 ± 1.03 %, which was less than 25 %. Hence the n value from the Korsmeyer Peppas model of release kinetics has been used to determine the swelling and diffusion mechanism of drug release (Gouda, Baishya, & Qing, 2017). The equation and the n value is as follows Korsmeyer Peppas Equation:
In the 0.1 N HCl media and along the with milk, the n value of all the 3 formulations (DC1, DC2, DC3) was found to be less than 0.5, which confirms that the release kinetics have followed Fickian diffusion (Supplementary Table III). In this case, the solvent diffusion is much greater than the process of polymeric chain relaxation. Fickian diffusion refers to the solute transport process in which the polymer relaxation time (tr) is much greater than the characteristic solvent diffusion time (td). In other media (water and PB pH-6.8), the values were slightly higher than 0.5, but less than 1.0 which shows that the formulations have followed non Fickian diffusion mechanism, in which tr ≈ td, the macroscopic drug release becomes anomalous (Fu & Kao, 2010).

3.2.8. In-vitro cytotoxicity and anti-HIV activity in human HIV cell lines

The CC50 value of pure DTG and DC1 formulation was far higher than their corresponding IC50 values (Fig. 9), which represented the effectiveness of the drug and nanoformulation with a high therapeutic index.
The cytotoxicity value (CC50) of DC1 formulation (195.28 ± 68.26 μg/mL) (Fig. 9 A) was far higher than the pure DTG (88.88 ± 5.10 μg/ mL) that evidenced the suitability of the biocompatible nature of the polymer chitosan.The IC50 value of pure DTG is 0.26 ± 0.08 ng/mL and the DC1 nanoparticles were 0.82 ± 0.71 ng/mL (Fig. 9 B). Although the IC50 value of the nanoparticles seem to be 2-fold higher than the pure drug, the ratio of DTG: Chn in the DC1 formulation is 1:1, which means 50 % drug and 50 % Chitosan. Hence, there was no substantial difference between the pure DTG and DTG-Chn nanoparticles in terms of its syncytium inhibition. This confirmed that the HIV-inhibiting activity of the DTG has not been affected after encapsulation inside the nanoparticles formulated using the biopolymer chitosan. With the obtained level of cytotoxic and inhibitory concentration, the selectivity index of the DC1 formulation was found to be broad (83010–2395818). This indicated that the isolated Chitosan could be suitable for encapsulating drugs and for adjusting dosage forms with a lower or higher ratio of the polymer, as required.

4. Discussions

The chitosan isolated from the novel crab species had shown an optimum yield of 40 % with the molecular weight ranging from 1200 − 1300 Da. A remarkable result of 93 % DDA was obtained by the chemical method of chitosan isolation, which could ultimately decide the purity and chemical properties of the sample. The higher deacetylation of chitin precursor had led to readily soluble chitosan in acidic medium (Islam, Bhuiyan, & Islam, 2017) and, hence offered better delivery of the DTG in the stomach pH media. The size analyses showed the minimum particle size of 140 nm for the optimized the DTG-Chn nanoparticles, which showed the compatibility of the spray drying process to produce nano-sized particles. The transformation of the rod-shaped pure DTG to spherical nanoparticles could be also confirmed by the SEM analysis post spray drying process. The electrostatic zeta potential surrounding the nanoparticles was highly positive, which explained the tendency of drug-loaded nanoparticles to permeate the biological membranes in vivo. Since the drug content of the nanoparticles is a crucial factor for effective dose fixing, the DC1 formulation showing greater than 75 %drug content was considered to be optimum. The chemical stability of the drug in the nanoformulation was confirmed by FTIR analysis, which also evidenced that the spray drying process in addition to polymer chitosan has not affected the chemical nature of the active drug molecule. The XRD profiles of the DTG-Chn nanoparticles have confirmed the solid-state transition of the drug from pure crystalline state to the amorphous state in the nanoparticles. The same kind of results was reported by other researchers as well (Cervera et al., 2011 and Nunthanid et al., 2004). Cervere et al., have obtained the spray-dried chitosan acid salts exhibiting the XRD characteristic peak at 20◦ (2θ) lower than the respective peak observed for reference pure chitosan, which indicated the amorphous transformation of the crystalline material after the spray drying process. The advantages of amorphous formulation have been explained by (Ambike, Mahadik, & Paradkar, 2005), wherein high internal energy and specific volume of the amorphous state materials can lead to enhanced dissolution and bioavailability, then the crystalline state. Another important factor to scale up the in vivo administration of the drug nanoparticles is the in vitro drug release profile. The DTG-Chn nanoparticles showed 100 % drug release in 0.1 N HCl media within a lesser time duration compared to the pure drug, which could improve the dissolution rate and overall bioavailability in vivo. The dramatic increase in drug release percentage can be explained by the reduction of particle size resulting in an increased specific surface area (Yang et al., 2012). Also, the solid-state transition of the pure DTG from the crystalline state to amorphous state after spray drying with Chitosan causes the increase in the drug release rate in the dissolution media. Belgamwar et al., have obtained the same result, where the dissolution of Dolutegravir has been substantially increased from 19.12 % to 54 % due to the reduction in crystallinity after encapsulation with HPβCD (Belgamwar, Khan, & Yeole, 2019). The particle size distribution of the developed DTG-Chn nanoparticles was found to be multimodal with high PDI value, which influenced the drug release pattern. The smaller sized particles were dissolved first, followed by the larger size particles (Rodrıguez, ́ Vila-Jato, & Torres, 1998) to exhibit a linear drug release profile. The MTT assay measures the viability of the cells, which directly depends on the level of metabolism in the live cells. The usage of HIV infected C1866 cell lines for measuring the cellular toxicity and the anti-HIV activity has been previously reported by Wang et al. Both the pure DTG and the DTG-Chn nanoparticles formulation were non-toxic till 3.2 μg/mL concentration, which provided evidence for lesser toxicity associated with the formulation. Further increase in the concentration of drugs and the nanoparticles caused a reduction in cell viability. The CC50 value of pure DTG and DC1 was 88.88 ± 5.10 μg/mL and 195.28 ± 68.26 μg/mL, respectively. The higher CC50 value associated with the chitosan formulation proved the biocompatibility of the polymer, which also indicated that a higher dose of the nanoformulation may be given without causing any cytotoxicity. The IC50 value of DTG and DC1was not significantly different, which represented that the anti-HIV activity of the drug has not been altered after processing into nanoparticles in addition to chitosan polymer.

5. Conclusion

The sequential extraction and complete structure elucidation of chitosan from the crab shell Portunus Sanguinolentus, the examination on its physicochemical properties and its application as drug delivery carrier has been explored successfully. The extracted chitosan possesses lower molecular weight, higher DDA % compared to the commercially available chitosan. Overall, our results validated that the identified biomaterial could be a novel source of the polymer due to its unique properties and also suggesting that the isolated chitosan may be used as an efficient excipient in the pharmaceutical industry. Dolutegravir was successfully processed as an optimized nanoparticle formulation using the extracted chitosan as a carrier, with the size distribution of 140 nm–548 nm, which is a remarkable result through the spray drying technique. The DTG has not been chemically degraded during the drying process that proved the compatibility of the technique for producing solid drug nanoparticles. The chemical stability and the crystallinity rearrangement of the DTG after the spray drying process were confirmed by FTIR and XRD analysis, respectively. A significant escalation in the surface area of the nanoparticles due to effective size reduction to the nanometer range favored the enhancement of the dissolution rate of the drug. Cell cytotoxicity assay using T-lymphatic cell lines by means of MTT assay and anti-HIV activity through syncytium inhibition assay evidenced that the selected DTG-Chn nanoparticles were less toxic compared to the pure drug. This research work evidenced the conversion of an anti-retroviral molecule into a nanoparticulate system with enhanced dissolution and minimum toxic effects, by encapsulating it inside a biopolymer. This chitosan-based nanoformulation in powder form would be highly compatible as milk/food admixture INS018-055  for paediatric HIV patients due to less difficulty in consuming the antiretroviral medicine, without compromising its therapeutic efficacy. The in-vivo study will be performed using mice models to analyze the biodistribution and enhancement in the bioavailability of the optimized nanoparticles and reported in near future.

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