INTRODUCTION:

Acquired Immune Deficiency Syndrome (AIDS) was first detected in 1981¹, and its causative organism human immunodeficiency virus (HIV) was discovered in the year 1983². But, till today, AIDS is a chronic and often fatal disease of pandemic proportions. HIV infection results in cell-mediated immune deficiency through progressive loss and dysregulation of CD4+ T lymphocytes, leading to AIDS³^,⁴^,⁵.

HIV not only infect CD4 lymphocyte, but also infects and injure both systemic and innate, central and peripheral nervous systems, immune systems through infection respectively of T-helper lymphocytes and of microglia, culminating in a wide spectrum of neuropsychiatric disorders or neuro AIDS. Neuro AIDS is associated with neurocognitive disorders such as HIV-associated dementia, minor neurocognitive disorder, mania/psychosis, anxiety, depression, seizures, mental slowness, forgetfulness, poor concentration, discombobulation, speech problem, decrease in spontaneity, myelopathy, and neuropathy with accompanying chronic neuropathic pain and physical disabilities⁶^,⁷^,⁸.

Brain/CNS is a very complex system; it provides a natural defense against toxic or infective agents circulating in the blood⁹^,¹⁰. The same mechanisms that protect the brain from foreign substances also restrict the entry of potentially active therapeutic moieties¹¹^,¹²^,¹³^,¹⁴^,¹⁵. The CNS delivery of antiHIV drugs is limited by the blood–brain and blood–CSF interfaces due to a combination of restricted paracellular movement, powerful metabolic enzymes and numerous transporters including members of the ATP-binding cassette (ABC) and solute carrier (SLC) superfamilies¹⁶. The BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain¹⁷. Tenofovir disoproxil fumarate (TDF), a prodrug of the nucleotide reverse transcriptase inhibitor, tenofovir (9-[9(R)-2-(phosphonomethoxy) propyl] adenine; PMPA). Tenofovir is an acyclic nucleotide analog with potent in vitro and in vivo antiretroviral activity. It was approved by USFDA for the treatment of HIV-AIDS in 2001. Tenofovir is primarily eliminated renally as unchanged drug with active tubular secretion by the kidney. The terminal half-life of tenofovir approximately 11-14 hours. TDF is a high water-soluble drug belonging to the class III of the Biopharmaceutics Classification System (BCS). The recommended adults oral dose of TDF is 300 mg per day. The oral bioavailability of tenofovir from TDF is 25%. The LD50 of TDF is 2.49 mol/kg. There is the negligible transport of TDF across the blood-brain barrier¹⁶^,¹⁸^,¹⁹^,²⁰. The concentration ration of the CSF and plasma is about 0.057 which is only 5% of plasma concentrations. This much concentrations is not effectively inhibit viral replication in the CSF¹⁹.

The blood–brain barrier (BBB) prevents drugs from permeability into the brain and limits the management of brain diseases. Intranasal delivery is a convenient route of drug administration that can bypass the BBB and lead to direct delivery of the drug to the brain due to the unique relationship between the nasal cavity and cranial cavity and transported via the olfactory epithelium and/or via the trigeminal nerves directly to the CNS²¹^,²². Indeed, drug accumulation in the brain following the intranasal application of a drug solution, or of a drug encapsulated in specialized delivery systems (DDSs), has been reported in numerous scientific publications²³^,²⁴^,²⁵^,. Various patents are also granted for delivery of bupropion, folic acid, mecamylamine, modafinil etc. to the brain through intranasal route²⁶^,²⁷^,²⁸^,²⁹.

In the literature it was found that many drugs like olanzapine³⁰, Leucine-enkephalin³¹, donepezil²⁵, doluxetine²⁷ etc. were successfully delivered into the brain through intranasal route. Pokharkar VB et al successfully developed a hybrid nanocarrier system based on lipid and polymer for the nasal delivery of TDF. An anomalous type (n > 0.5) of drug release pattern was observed with potential to transport TDF across the nasal mucosa with an average flux of 135.36 μg/cm2/h³². Zhang T et al developed a TDF loaded pH-Responsive Nanoparticles prepared from the blend of poly (lactic-co-glycolic acid) (PLGA) and methacrylic acid copolymer (Eudragit® S-100, or S-100) for the prevention of HIV transmission via intravaginal administration. There was a 4-fold increase in the drug release rate from 75% S-100 blend in the presence of semen fluid simulant over 72 h³³. Jayant RD et al developed a novel approach for the co-encapsulation of an anti-HIV drug (tenofovir) and a latency-breaking agent (vorinostat), using magnetically guided layer-by-layer (LbL) assembled nanocarriers for the treatment of neuroAIDS³⁴. The major drawbacks of these formulations are larger particle size, poor brain target ability, toxicity, instability, drug loss on storage, expensive, no sustained release properties inside the brain, and lack of commercial viability.

Colloidal drug carriers offer a number of potential advantages for drug delivery. Lipid nanoparticles (LNPs) have attracted special interest during the last few decades. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) are two major types of Lipid-based nanoparticles. SLNs were developed to overcome the limitations of other lipidic nanocarriers, such as emulsions, liposomes, and polymeric nanoparticles because they have advantages like good release profile and targeted drug delivery with excellent physical stability. In the next generation of the lipid nanoparticle, the presence of solid cum liquid lipid in the NLC leads to greater drug encapsulation and loading and long-term colloidal stability. The conception of NLC that is lipid matrices which are solid, but not crystalline is derived from the fact that the crystallization process itself causes the expulsion of the drug. By using special mixtures of solid lipids and liquid lipids, the particles become solid after cooling but do not crystallize. NLC has easily stabilized with a minimum possible concentration of surfactants along with best results of stability, entrapment, and release. Sometimes, even 0.5-1% of the surfactant is sufficient for developing stable NLC³⁵^,³⁶^,³⁷^,³⁸.

Compretol 888 ATO is chemically Glyceryl behenate an ester of glycerin with behenic acid. Oleic acid consists chiefly of (Z)-9-octadecenoic acid together with varying amounts of saturated and other unsaturated acids. Both Compritol 888 ATO and oleic acid both are found in various dietary foods. These are included in the FDA Inactive Ingredients and are generally regarded as a relative nonirritant and nontoxic material. These are used in cosmetics, foods, and pharmaceutical formulations³⁹^,⁴⁰^,⁴¹. Both of these are fatty acid and degraded by oxidation in vivo and form acetyl-CoA (coenzyme A) which is the entry molecule of the citric acid cycle for energy production. So NLC formulation with these two materials will be biocompatible and biodegradable, stable and can be easily scaled up as compared to other lipid formulations.

The present investigation was aimed to evaluate the developed TDF loaded NLC system to have the potential of brain delivery via the non-invasive intranasal route (IN). TDF loaded NLCs were tested for its physicochemical characteristics, cell viability, nasal toxicity, in vitro drug release as well as ex-vivo nasal permeation, and in vivo brain distribution.

MATERIAL AND METHODS:

Chemicals and reagents:

TDF and Compritol ATO 888 were received as a gift sample from Micro Labs Ltd. (Bangalore, India). Oleic acid, Pluronic F-68 and Tween 80 were purchased form HiMedia (Mumbai, India). Ethanol was purchased from Merck Millipore, Mumbai, India. Urethane, phosphate buffer saline, pH 7.4 and DMEM were purchased from HiMedia, Mumbai, India. bEnd.3 cell lines were obtained from ATCC. MTT reagent and EDTA were purchased from HiMedia (Mumbai, India). All other chemicals and reagents were of highly purified grade and were used without further purification.

Preparation and physicochemical evaluation of NLCs:

The details of the preparation method were published in our previous paper⁴². In brief The NLCs were prepared by modified emulsion solvent diffusion method. A Response Surface Central Composite Design (CCD) was applied to investigate the effect of formulation variables on dependent variables and statistically optimize the formulation factors using Design-Expert software (Version 10.0.1, Stat-Ease Inc. USA)⁴³^,⁴⁴. The Lipid to Drug ratio, Aqueous phase pH and Sonication time (min) were selected as the factors and were accordingly varied. While Particle size (nm), PDI and %EE were selected as dependent variables/responses. The optimized NLCs were further investigated for the effect of different surfactant and concentrations on its particle size, zeta potential, PDI and %EE. Among numerus trials two NLC formulations (T4 and T5) were selected for further ex-vivo and in-vitro studies. The compositions of T4 and T5 are listed below (Table 1).

Table 1: Composition of T4 and T5

Formulation	Lipid drug ratio	Tween 80+Pluronic F68 (1:1) (%w/w)
T4	2.18: 1	1.5
T5	2.18: 1	1.0

The NLCs were characterized for Particle size, PDI using dynamic light scattering (DLS) technique. while zeta potential was determined by Electophoretic Light Scattering (ELS) technique. Percent encapsulation efficiency was determined by separating the unentrapped TDF from TDF-NLCs by using Sephadex G-25 column. The incompatibility of the ingredients of the formulation was checked by the Fourier Transform Infrared (FT-IR) spectroscopy, X-ray diffractometry (XRD) and Differential scanning calorimetry (DSC). The shape and surface characteristics of NLCs were determined by Scanning electron microscopy (SEM). The morphology of NLCs was observed under a transmission electron microscope (TEM). The details have been published in our previous paper⁴².

Cell viability study:

Cell culture:

The bEnd.3 cerebral cortex cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were seeded into cell culture dishes containing DMEM supplemented with 10% new calf serum, l-glutamine (5 mmol/L), non-essential amino acids (5 mmol/L), penicillin (100 U/mL), and streptomycin (100 U/mL) at 37ºC in a humidified 5% CO₂ atmosphere.

In vitro cellular cytotoxicity assays:

Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay. bEnd.3 cells were plated in 96-well plates with 100µL medium at a density of 1x10⁴ cells per well. After 12 h, cells were exposed to various concentrations of T4, T5, TDF and blank NLCs for 24 and 72 h. Cells treated with DMEM and 10% DMSO was considered as negative control and positive control respectively. MTT solution was directly added to the media in each well, with a final concentration of 0.5mg/mL and incubated for 4 h at 37ºC. The resulting formazan crystals were solubilized with 150µL DMSO. The absorbance was measured using an enzyme-linked immunosorbent assay reader at 570nm, with the absorbance at 630nm as the background correction. The effect on cell proliferation was expressed as the percent cell viability. Untreated cells were taken as 100% viable.

Ex vivo estimation of nasal toxicity in pig nasal mucosal membrane:

Histopathological studies on isolated pig nasal mucosa were conducted to assess the possible local toxic effects following nasal instillation of TDF loaded (1 mg/ml) NLCs⁴⁵.

Preparation of isolated pig nasal mucosal membrane:

The nasal-cavity mucosa of a one-year old pig, was obtained from the local slaughter house. Within 15 min of the sacrifice of the animal, the nasal cavity was fully exposed by a longitudinal incision through the lateral wall of the nose while avoiding the damage of the septum. Following, the mucosa was carefully removed and immediately immersed in 900ml of ice-cold Ringer’s solution⁴⁶.

Application of treatments:

Two segments were carefully separated from the anterior and the posterior regions of the mucosa in the nasal cavity. Each segment was sectioned into three pieces. The pieces were treated with TDF loaded NLCs, phosphate buffer saline pH 6.4 (negative control) and isopropyl alcohol (positive control), respectively. After treatment for 1, 2, 4 and 8 h, the pieces were washed with distilled water and preserved in 10% formalin solution for 48 h⁴⁷.

Histopathological studies:

Briefly, the samples were dehydrated by treatment with serial dilutions of methyl alcohol, ethyl alcohol, and absolute ethyl alcohol, respectively. Specimens were cleared in xylene embedded in paraffin in a hot air oven. The temperature of the oven was adjusted at 56ºC and the samples were kept for 24 h. Paraffin-beeswax tissue blocks were sectioned by a sledge microtome (Leica Microsystems SM2400, Cambridge, England). The obtained tissue sections (3–4µm thickness) were collected, deparaffinized, stained by hematoxylin and eosin, and examined under a light microscope⁴⁸.

Ex vivo nasal permeation studies:

Nasal diffusion was carried out using Franz diffusion cell with a receptor volume capacity of 20ml using pig nasal mucosa membrane as a dialyzing membrane. The freshly excised pig nasal mucosal membrane was rinsed thoroughly with phosphate buffer saline (PBS) pH 6.4 from which superior nasal membrane were identified. Nasal mucosa having a thickness of 0.3mm was mounted on the diffusion cell with the mucosal surface facing donor compartment and the serosal surface facing receptor chamber. Comparative nasal diffusion study was performed in triplicate by taking TDF loaded NLCs and TDF solution. Receptor chamber was filled with PBS pH 6.4 and was allowed to stir continuously using magnetic bead in a manner that PBS touches the serosal surface of the mucosa. The temperature in the receptor chamber was controlled at 37±1◦C using a circulating water bath. Aliquots of 1 ml were withdrawn at different time intervals (0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24 and 48 h) and replaced with an equal volume of PBS. Samples were analyzed for drug content by UV spectrophotometer at 260nm.

Biodistribution and pharmacokinetic studies in rats:

Animal handling and drug administration:

The protocol for the animal study was approved by the Institutional Animal Ethical Committee (IAEC) of Dibrugarh University, Dibrugarh, Assam, India (Approval No. IAEC/DU/83 dated 27/03/2015). Albino Wistar rats weighing between 180 and 250g, procured from Chakraborty Enterprise, Kolkata, India were selected for the biodistribution study. Rates were acclimatized with the laboratory environment for 21 days prior to the commencement of animal study. Animals were divided into six prime groups (A, B, C, D, E and F) each consisting of 15 animals. Further they were again subdivided into five subgroups (1, 2, 4, 8, and 24 h) each containing three animals. Animals were marked on the tail for proper identification. The sub grouped animals were kept in a cages (55 × 32.7×19 cm) made up of polypropylene, over a bed of paddy husk in an air and temperature controlled environment (23 ± 2 °C). Animals were fed with laboratory animal food pellets and water.

Before the experiment, animals were anesthetized by injecting urethane (1.2g/kg B.W.) through intraperitoneal route. First two groups (A and B) received T4 and T5 through IN route, second two groups (C and D) received T4 and T5 through IV route and last two groups (E and F) received TDF through both IN and IV route equivalent to 5mg/kg dose of TDF respectively. For IN administration groups, formulations equivalent to 1 mg/ml of TDF were prepared and 40µL of the formulation was administered into each nostril of anaesthetized rats. Rats were held from back in slanted position during IN administration. Similarly, for IV administration group, 200µL of formulations (1mg/ml of TDF) was injected through the rat tail vein. At each of the following time points:1, 2, 4, 8 and 24 h (n = 3, each time point), blood samples were collected from the heart into heparinized tubes. Rats were then sacrificed by cervical dislocation method. The brain blood vessels were perfused with 0.9% w/v sterile physiological saline solution through the common carotid artery to remove blood⁴⁹^,⁵⁰. Perfusing the brains assures that no TDF is remained in the blood vessels of the brain as free drug or in the NLC formulation and the drug concentration in the brain shown by the analytical procedures are only the amount of TDF that has actually crossed the BBB. The rat brains were then collected by opening the cranium and kept in 15 mL centrifuge tubes after a thorough wash of the brain with 0.9% w/v sterile physiological saline solution. Just after collection of blood samples, they were centrifuged at 5000 rpm for 15 min at 4°C to separate plasma from blood cells. Separated plasma was collected in Eppendorf tubes and kept in -20°C until further use. The brains were also kept in -20°C until further use.

Extraction of TDF from blood and brain samples:

Plasma proteins were precipitated by mixing 1mL Acetonitrile with 100μL of plasma sample followed by centrifugation at 13,000rpm for 15 min at 4ºC. The supernatant was collected in a tube. This process was repeated thrice with the residue. The supernatants were combined and evaporated to dryness at 45°C in a hot-air oven. It dried residue was reconstituted in 100μL of HPLC mobile phase and 20μL was injected into the HPLC column for the detection of the TDF (𝜆_max 260 nm)⁵¹. Collected rat brains were homogenized with 10 mL deionized water in a tissue homogenizer and 100μL of homogenate was taken in 2mL Eppendorf tube. Tissue proteins were precipitated by mixing with 1mL of acetonitrile followed by centrifugation at 13,000rpm for 15 min at 4ºC. The supernatant was collected. This process was repeated thrice with the residue and the supernatants were finally combined and evaporated at 45°C in a hot-air oven. The dried residue was reconstituted with 100μL of HPLC mobile phase and 20 μL was injected into the column for the detection of TDF (𝜆_max 260 nm).

Chromatographic Conditions:

An RP-HPLC system (Agilent Technologies, 1260 Infinity HPLC, California; USA) was utilized to quantify TDF in plasma and brain samples. The HPLC system was equipped with photodiode array detector set at 260 nm and Agilent Technologies mRP-C18 column (250 mm × 4.60 mm, ID; 5μm particle size) associated with a guard column (50mm × 4.60mm, ID; 5μm particle size). TDF was eluted by isocratic flow at a rate of 1 mL/min at ambient temperature, with a mobile phase comprising a mixture of Acetonitrile and Water (70:30) pH adjusted to 4.5 with ortho-phosphoric acid (OPA).

Standard Solutions:

A stock solution of 1.0mg/mL was prepared by dissolving TDF in HPLC mobile phase at room temperature. Serial dilutions of the stock solutions were made, and 20μL was introduced into the HPLC column to prepare the calibration curve. The calibration curve for TDF was prepared with seven concentrations: 1.6, 3.2, 6.4, 12.8, 25.6, 51.2 and 100µg/mL. Stock and working standard solutions were protected from light and stored at −20°C until used⁵²^,⁵³.

Assay Validation:

To validate the HPLC method, the following parameters were investigated: selectivity, linearity, precision and accuracy, the limit of quantification (LOQ), and the limit of detection (LOD).

Selectivity:

Selectivity indicates the lack of interfering peaks at the retention times of the assayed drug. The specificity of the method was determined by comparing the chromatograms obtained from the samples containing TDF with those obtained from blank plasma and brain samples.

Recovery and Linearity:

In the analysis of TDF in rat plasma, the analytical recovery of TDF was determined at concentrations of 1.6, 12.8, and 100µg/mL (n = 3). Samples of the plasma and brain tissue without drug were spiked with known amounts of the drug to achieve the specified concentration. These samples were processed by the analytical method described above and peak areas were compared to that obtained by direct injection of the drug with the mobile phase, that is, the standard curve data. To calculate linearity, calibration curves were constructed by linear regression within the range of 1.6–100µg/mL of TDF, using seven standard solutions. The limit of quantification (LOQ) was determined as the lower value in the calibration curve. For the LOD, three samples at concentrations near to the smallest concentration of the standard curve (triplicate) were analyzed in order to obtain the standard deviations.

Precision and Accuracy:

Precision was determined by the coefficient of variation (CV) and accuracy as the percent relative error (RE). Intraday precision and accuracy data were obtained by analyzing aliquots of plasma samples at low (1.6 µg/mL), medium (12.8µg/mL), and high (100µg/mL) levels of the TDF concentration (n = 3). Inter-day reproducibility was determined over 3 days.

Pharmacokinetic analysis:

Confocal imaging of Rat Brain cryosection:

To confirm the BBB crossing ability of the prepared NLCs, ex-vivo imaging of rat brain cryosections were performed using the confocal laser scanning microscope (CLSM). Coumarin 6 (C6)-labeled NLCs were prepared using the same method as described earlier in the section ‘Preparation of NLCs’ with minor modification. Briefly C6 was first dissolved in absolute ethanol (1mg/mL), added to the lipid ethanol mixture and mixed well. The rest of the formulation process was unchanged. C6 loaded NLCs showed the similar physicochemical properties of NLCs like particle size and zeta potential. The C6 loaded NLCs were injected through intranasal route in healthy rats. Then, rats were sacrificed at 1, 2, 4, 8, and 24 h and brains were collected, washed thoroughly with PBS and were stored at −20°C until further use. The brain tissues were sliced into sections (6–10μm in thickness) using a Shandon Cryotome E (Thermo Electron Corporation, USA) and then stained with DAPI (1μg/mL) for 2 min, washed three times with PBS pH 7.4. The stained tissues were mounted on microscopy slides before analysis by CLSM. The brain tissues were imaged under a confocal laser scanning microscope (TCS SP8, Leica, Germany) and fluorescent microscope using the wavelengths for DAPI (Ex./Em. = 358/461 nm) and C6 (Ex./Em. = 465/505 nm)⁵⁴^,⁵⁵.

Statistical Analysis:

All pharmacokinetic parameters of TDF solution and NLC formulations were expressed as the mean ± standard deviation (SD). The data were analyzed with Graph Pad Prism (Version 5.03, Copyright 2017, GraphPad Software, Inc.; USA). One-way ANOVA study was done to determine the significant difference between the formulations and TDF solution. The significance of the difference between TDF solution and NLC formulation data was determined with Student’s t-test. A statistical difference p > 0.05 was considered significant.

RESULT AND DISCUSSION:

Evaluation of NLCs:

The particle size of the optimized NLCs T4 and T5 were found to be 94.7±15.70nm with PDI of 0.380±0.024 and 134.3±9.71nm with PDI of 0.358±0.038 respectively. The developed NLCs, were small enough to cross the BBB. Previous literature shows that nanocarriers having a particle size below 150nm cross the BBB and get into the brain very easily. The zeta potential value of -17.± 3.87mV and -17.17±1.05mV was found for T4 and T5 respectively with %EE of 35.5±1.04 and 34.2±2.78 (Table 2). NLCs were found to be spherical in shape with uniform distribution. SEM and TEM analysis revealed that the NLCs had a smooth surface and did not show any aggregation (Figure 1). The FTIR, DSC and XRD study confirms the compatibility among the ingredients of the formulation. The in vitro drug release study of the developed NLCs in aCSF showed slow release profile of TDF. The percentage of drug released from the different NLC formulations (T4 and T5) was 60% at 48 h.

Figure 1: TEM image of T4 (a), T5 (b) and SEM image of T4 (c), T5 (d)

Ex vivo nasal permeation studies:

Figure 4 shows permeation profile of drug from NLCs (T4 and T5) and plain drug solution across nasal mucosal membrane. TDF permeated very slowly both from T5 (57.62%±0.59 in 48 h) and T4 (57.19%±0.96 in 48 h), whereas permeation was found to be increased from TDF solution (100% before 10 h). This slow permeation of TDF from NLCs could be attributed firstly to the sustained release behavior of nanosized delivery system. Secondly, NLCs with Tween 80 is less negatively charged which interacts with negatively charged sites on mucosal epithelial cell linings and sialic acid present in the mucin which transiently open tight junctions of mucosal membrane by translocating proteins ZO-1 and occludin which controls membrane tightness and diffusion through intercellular spaces. Drug permeation was correlated with the drug release from the NLCs.

Figure 4: % of TDF permeated through pig nasal mucosal membrane

Biodistribution and pharmacokinetic studies in rats:

The reversed-phase HPLC-UV method described validated, and used for TDF quantification, provides great sensitivity and specificity, and high sample throughput required for pharmacokinetic studies. The chromatograms showed a good baseline separation and the mobile phase used resulted in optimal separation. The method was selective for TDF since it shows that no interfering peaks appeared near the retention time of the compound of interest (Figure 5). The LOQ (0.438 µg/ml) and LOD (0.131 µg/ml) values were low, indicating the good sensitivity of this HPLC method.

The precision of the applied analytical method was very accurate. The observed value of precision for different concentrations ranged from 2.12 to 5.27%. The observed accuracy value varied from 95.83 to 122.5%. The accuracy of intra and inter day analysis varied between 98.19 – 123.29% and 98.19 – 123.29%, respectively. The precision of intra and inter day analysis varied between 2.12–5.27% and 2.12–5.27%, respectively.

The sample preparation used in this study involved only a single step (i.e., deproteinization with acetonitrile). This condition was optimal for sample preparation as it resulted in clean chromatograms.

TDF level was assessed in CSF after intranasal administration of T4 and T5 to determine the efficacy of the formulation in delivering the therapeutic concentration in brain. The mean CSF concentration–time profile after i.n. administration of T4 and T5 are represented in Figure 6.

The maximum concentration of TDF appeared in CSF was 0.4 ± 0.16 µg/mL after i.v. administration and 1.2 ± 0.77 µg/mL after intranasal administration of T5 at 120 ± 0.00 min. While 0.4 ± 0.16 µg/mL and 0.5 ± 0.5 µg/mL concentration in CSF was found afetr i.v. and intranasal administration of T4 at 120 ± 0.00 min respectively.

The rapid appearance of C_max in CSF after intranasal administration of T5 might be because of extracellular transport of TDF which involves transport via perineural space present surrounding olfactory axon loosely adherent to perineural epithelium or through epithelial cell junction. Transport may occur via tight junctions or clefts in the epithelium into the CSF as they are directly connected anatomically in submucosa and subarachnoid extensions, perineural space surrounding olfactory nerves as they penetrate the cribriform plate.

The AUC_0–24of TDF in CSF post intranasal administration of NLCs was 4-fold greater than AUC_0-24after i.v. administration of T5 NLCs (96.32 ± 1.24 µg/mL.h vs. 26.85 ± 0.86 µg/mL.h). CNS uptake of TDF was significantly (p<0.05) high for intranasal NLCs compared to i.v. solution. There are several reports so far in the literature that TDF has very limited access in the CNS after oral administration⁵⁶^,¹⁹. Therefore, these results warrant promise in treating neuro-AIDS for nose to brain targeting of TDF.

The drug transport from nasal cavity to the CNS involves combination of pathways such as olfactory, trigeminal and systemic. Tween 80 present in the NLCs might have contributed in opening tight junctions between sustentacular cells (or clefts) and olfactory neurons, TDF may have been diffused passively and via olfactory neuronal cell by endocytosis or pinocytosis and transported by axons to the olfactory bulb. When a drug is absorbed from the respiratory epithelia, it reaches the systemic circulation before crossing the BBB. However, TDF is highly bound to plasma proteins, thus transport through systemic route would be negligible.

The t_1/2T5 NLCsfor i.v. administration in plasma was found to be 10.11 h and in CNS it was 20.3 h, while in intranasal administration of T5 NLCs plasma t_1/2was 10.91 h and 13.17 h in CNS. TDF on oral administration has very long half-life of 17 h after single dosing. The enhanced CNS access of the drug was observed which may be attributed to various factors among them is significant increment in the solubility of this hydrophobic drug with lipid used in the formulation.

Figure 5: (A) HPLC chromatogram of pure TDF. HPLC Chromatograms demonstrating selectivity with (B) blank rat brain; (C) Rat brain post administration of T4 and (D) Rat brain post administration of T5 through intranasal route.

Figure 6: Brain pharmacokinetic profile of T4 and T5 post intranasal administration

Table 3: rat plasma and brain pharmacokinetic parameters for intravenous bolus TDF solution, T4 and T5

Parameters	Plasma pharmacokinetic profile			Brain Pharmacokinetic profile
Parameters	TDF solution	T4	T5	TDF solution	T4	T5
C_max (µg/mL)	0.32 ± 0.002	0.41 ± 0.001	0.41 ± 0.001	0.19 ± 0.001	0.39 ± 0.003	0.35 ± 0.002
T_max (h)	1	8	8	8	2	2
AUC_0−t (µg-h/mL)	21.47 ± 0.68	29 ± 0.72	33.2 ± 0.63	8 ± 0.2	27.53 ± 0.66	26.85 ± 0.84
t_1/2(h)	13.89 ± 0.68	8 ± 0.08	10.11 ± 0.62	10 ± 0.56	12.08 ± 0.98	20.3 ± 0.93
MRT (h)	65.39 ± 0.88	32 ± 1.12	42.3 ± 0.77	12 ± 0.59	76.64 ± 0.68	74.11 ± 0.45

Table 4: Rat plasma and brain pharmacokinetic parameters for intranasal bolus TDF solution, T4 and T5

Parameters	Plasma pharmacokinetic profile			Brain Pharmacokinetic profile
Parameters	TDF solution	T4	T5	TDF solution	T4	T5
C_max(µg/mL)	0.33 ± 0.001	0.32 ± 0.003	0.4 ± 0.002	0.17 ± 0.001	0.47 ± 0.005	1.11 ± 0.005
T_max (h)	24	2	1	1	2	2
AUC_0−t(µg-h/mL)	22.3 ± 0.48	50.94 ± 0.78	53.64 ± 0.76	23.92 ± 0.48	54.67 ± 0.84	96.32 ± 1.24
t_1/2(h)	22 ± 0.54	12.49 ± 0.32	10.91 ± 0.58	12 ± 0.61	17 ± 0.86	13.17 ± 0.44
MRT (h)	28 ± 0.36	16.34 ± 0.88	26.14 ± 0.98	13.7 ± 0.22	55.62 ± 0.82	44.7 ± 0.94

CONCLUSION:

The TDF loaded NLCs were successfully developed by modified emulsion solvent diffusion method for effective brain delivery of TDF. The developed NLCs were the best stable in refrigerated condition and safe for intranasal administration. The NLCs showed sustained release profile of TDF in a CSF and in vivo in rat brain. The cytotoxicity study on bEnd.3 cell line and histopathological study on pig nasal mucosa reveal the nontoxic profile of the NLCs. The in vivo plasma and brain pharmacokinetic investigation in the rat model revealed that NLCs rapidly reached the brain and yielded higher MRT, C_max, and AUC in rat brain compared to those data with TDF solution. The rat brain pharmacokinetic data and CLSM imaging of rat brain cryosections confirm that the developed NLCs had effectively crossed the BBB delivering TDF in a sustained manner for a prolonged period of time in the brain. This may provide an effective therapeutic strategy to combat the challenges of HIV infection in the brain. Further, preclinical and clinical development studies are warranted.

ACKNOWLEDGMENTS:

The authors gratefully acknowledge the experimental/ analytical support of Guwahati Biotech Park, Technology Complex, IIT Guwahati, The Sophisticated Analytical Instrument Facility (SAIF), IASST, Guwahati, Tezpur University, Tezpur and College of Veterinary Science, Guwahati. This work was financially supported by the Department of Science and Technology, Ministry of Science and Technology, Government of India.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 22.12.2019 Modified on 18.02.2020

Research J. Pharm. and Tech. 2020; 13(11):5411-5424.

DOI: 10.5958/0974-360X.2020.00946.4

Trial	Particle size (nm)	PDI	%EE	Zeta potential (mV)	%DL
T4	94.7 ± 15.70	0.380 ± 0.024	35.5 ± 1.04	-17.0 ± 3.87	1.22 ± 1.12
T5	134.3 ± 9.71	0.358 ± 0.038	34.2 ± 2.78	-17.17 ± 1.05	1.77 ± 0.89