Accepted Manuscript

Title: Solid Lipid Nanoparticles (SLN) of Efavirenz as lymph

targeting drug delivery system: Elucidation of mechanism of
uptake using chylomicron flow blocking approach

Author: Vivek Makwana Rashmi Jain Komal Patel Manish

Nivsarkar Amita Joshi

PII: S0378-5173(15)30212-X
DOI: http://dx.doi.org/doi:10.1016/j.ijpharm.2015.09.014
Reference: IJP 15199

To appear in: International Journal of Pharmaceutics

Received date: 23-6-2015

Revised date: 2-9-2015
Accepted date: 8-9-2015

Please cite this article as: Makwana, Vivek, Jain, Rashmi, Patel, Komal,
Nivsarkar, Manish, Joshi, Amita, Solid Lipid Nanoparticles (SLN) of Efavirenz
as lymph targeting drug delivery system: Elucidation of mechanism of uptake
using chylomicron flow blocking approach.International Journal of Pharmaceutics
http://dx.doi.org/10.1016/j.ijpharm.2015.09.014

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Solid Lipid Nanoparticles (SLN) of Efavirenz as lymph targeting drug delivery system:
Elucidation of mechanism of uptake using chylomicron flow blocking approach

Vivek Makwana1, Rashmi Jain1, Komal Patel1, Manish Nivsarkar2, Amita Joshi2#

1. National Institute of Pharmaceutical Education and Research (NIPER) - Gandhinagar, c/o

B. V. Patel PERD Centre, Thaltej, S.G. Highway, Ahmedabad
2. B. V. Patel PERD Centre- Thaltej, SG Highway, Ahmedabad

Corresponding author#
Dr. Amita Joshi
Scientist-B, PERD Centre and Adjunct Faculty, NIPER-G
NIPER-G, Department of Pharmaceutics
c/o B.V. Patel PERD Centre,
S.G. Highway, Thaltej, Ahmedabad- 380054
Tel.: +91-79-27439375/ 27416409
Fax: +91-79-27450449
Email- amitagarg4@gmail.com

Graphical abstract
Abstract
The aim of the present work was to develop a lymph targeted SLN formulation of antiretroviral

(ARV) drug and to have an understanding of its underlying mechanism of uptake by the

lymphatics. The lymphatics are the inaccessible reservoirs of HIV in human body. Efavirenz

(EFV) is a BCS class II, ARV drug that undergoes extensive first pass metabolism. The EFV SLN

formulation was prepared using Gelucire 44/14, Compritol 888 ATO, Lipoid S 75 and Poloxamer

188 by hot homogenization technique followed by ultrasonication method, with mean particle

size of 168nm, polydispersity index (PDI) < 0.220, and mean zeta potential of -35.55 mV. DSC

and XRPD studies revealed change in cyrstallinity index of drug when incorporated into SLN. In

vitro drug release was found to be prolonged and biphasic in PBS pH 6.8. There was no significant

change in the mean particle size, PDI, zeta potential and entrapment efficiency of EFV SLN after

1
storage at 30 ± 2°C/60 ± 5%RH for two months. The results from lymphatic transport and tissue

distribution study indicate that a significant part of the EFV had by-passed portal system and was

recovered in the lymph via chylomicron uptake mechanism. Reduction in the amount (44.70%)

of the EFV reaching to liver indicates that major amount of EFV bypasses the liver and thereby,

enhances the oral bioavailability of the EFV. A significant amount of EFV was found in spleen,

a major lymphatic organ. EFV SLN seems to have potential to target the ARV to lymphatics for

the better management of HIV.

Keywords: Anti-retroviral therapy, HIV, lymph targeting, Colloidal drug delivery, Particulate
delivery

1. Introduction

Today, antiretroviral (ARV) drugs are the major thrust for effective management of Human

Immunodeficiency Virus (HIV) and controlling the progress of Acquired Immune Deficiency

Syndrome (AIDS) and its symptoms. However, an effective cure for HIV infection is still a dream

for scientists and current strategies are not enough, urging for the search of novel and improved

options (Gupta and Jain, 2010).

HIV is localized in certain inaccessible compartments of the body such as the Central nervous

system (CNS), and the lymphatic system, wherein the majority of drugs cannot be maintained for

the necessary duration and in the therapeutic concentrations required. HIV mainly resides and

perpetuate in the anatomical (CNS, lymphatics, and the genitals) and cellular reservoir (CD+T

lymphocytes and monocytes / macrophages) of the human body. However, majority of the

antiretroviral drugs from the conventional delivery system are unable to reach to these

inaccessible HIV reservoirs (Alex et al., 2011). Also, minimum effective concentration of drug

cannot be maintained for the necessary duration of action in these HIV reservoirs. Therefore,

targeting to lymphatics that serve as viral sanctuaries confers therapeutic advantage and seems to

be a logical approach.

2
The essential requirement for the lymphatic uptake includes, system should have a log P around

3-5, medium and long chain triglycerides solubility of more than 50mg/ml and a size of around

100 nm (Bandyopadhyay et al., 2012; Dane et al., 2011). In this framework, development of lipid

based nanoformulations of ARVs to improve the effectiveness of therapy by targeting lymphatics

holds promise for the better management of the disease. This will ensure the availability of ARV

drugs at the viral sanctuaries i.e., lymphatics, within the theraputic window and for a prolonged

period. Drugs that are lymphatically, rather than portally transported, avoids first-pass

metabolism by the liver. Also, since these formulations bypasses portal circulation, their

bioavailability is also not affected by the concurrent administration of anti-tubercular drugs like

Rifampicin.

Solid lipid nanoaparticles (SLNs) are the class of particulate drug carriers made from lipids that

remain in the solid state at room and body temperature (Trevaskis, et al., 2008). SLNs combine

the advantages and avoid the disadvantages of other colloidal carriers (Mehnert and Muller,

2001), such as possibility of controlled drug release and targeting, increased drug stability and

payload, incorporation of hydrophilic and lipophilic drugs, avoidance of organic solvents and

biotoxicity to the carrier as well as bypassing the liver. SLNs can induce stimulation of

chylomicron formation by enterocytes which promote the absorption of lipid matrix through

intestinal lymphatics (Hu et al., 2004). In the present study, the potential of orally, administered

Efavirenz (EFV) loaded SLNs for the lymphatic uptake system has been examined. Also, an

attempt has been made to elucidate the mechanism of lymphatic uptake, using chylomicron flow

blocking approach. EFV is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the

treatment of HIV-1 infection. EFV is recommended as a part of HAART for the treatment of

HIV/AIDS in WHO regimens. EFV is a BCS class II drug having poor aqueous solubility and

high permeability. EFV undergoes extensive first pass metabolism leading to low and erratic

bioavailability.

3
2. Material

Compritol 888 ATO (glyceryl behenate) and Gelucire 44/14 was obtained as a gift sample from

Gattefosse Mumbai, India. Efavirenz was kindly supplied by Mylan Pharmaceuticals Inc.,

Hydrabad, India, as a gift sample. Poloxamer 188 and Mannitol were purchased from Sigma–

Aldrich (Bangalore, India). Soya lecithin was received as a gift sample from Phospholipid GmbH,

Germany. Purified water obtained from a Milli Q Plus (Millipore) was used for all experiments.

All other chemicals and reagents were of analytical grade and used without further purification.

3. Methods

3.1Solubility study of EFV in lipids and surfactants

Fixed amount of each solid lipid/surfactant (10 mg) was taken in a test tube and was heated above

the melting point of the lipids, at 70°C, on a water bath (Equitron Chennai India). To the melted

lipid/surfactant, 10 mg of EFV was added and vortexed. In case, EFV was not dissolved, the

amount of lipid/surfactant was increased in the increments of 10 mg, untill the entire EFV was

solubilized. The amount of solid lipid/surfactant required to dissolve 10 mg of EFV was noted

(Li et al., 2008). Various solid lipids such as Gelleol pellets, Gelleol pastilles, Imwitor 900,

Precirol ATO, Compritol 888 ATO (COM), Compritol E ATO, Gelucire 44/14 (GEL), Gelucire

43/011, Gelucire 50/13, softisan 142 and Dynasan 114 were screened for solubility of EFV.

While, surfactants and cosurfactants screened included Poloxamer 188, Poloxamer 407, Labrafil

M2130, Cremophore 40, Cremophore EL, Lipoid S 75.

3.2 Preparation of EFV-SLN

EFV SLNs were prepared using hot homogenization process followed by ultrasonication.

Formulation was prepared using GEL and COM as solid lipids and Lipoid S 75 and Poloxamer

188 as Surfactant and Co-surfactant, respectively. EFV, GEL, COM and Lipoid S 75 were

dissolved in 10 ml of chloroform. Organic solvent was completely removed by heating on water

bath at 75º- 80ºC. Drug embedded lipid layer was melted by heating at 5°C above melting point

4
of the lipid. The aqueous phase was prepared by dissolving Poloxamer 188 in Milli-Q water and

heated to same temperature as that of the molten lipid phase. Hot aqueous phase was then added

to the molten lipid phase and homogenization was carried out with the help of High Speed

Homogenizer (HSH) (Polytron PT1600 E Kinematica, Switzerland) for 7 min. at 15000 rpm.

Coarse hot, oil in water emulsion was ultrasonicated using probe sonicator (Sonic prepultra

homogenizer, Germany) (Manjunath and Venkateswarlu, 2005). The formed emulsion was

dispersed in cold water (0–5ºC) under stirring. The effect of various process variables like,

homogenization time, homogenization speed, sonication time, on particle size and PDI was

studied and optimized (Table 1)

3.3 Freeze drying of EFV-SLNs

The EFV SLN suspension was centrifuged at 12,000 rpm and the supernatant was discarded. The

pellet was washed with Mill-Q water. Milli-Q water (10 ml) containing cryoprotectant was used

for reconstituting the pellet. The dispersion of EFV SLNs with cryoprotectant was kept at -80°C

for 2h and subsequently freeze dried in lyophiliser (Allied Frost Pvt. Ltd., New Delhi, India) for

48h at temperature of -80°C and vacuum of 0.370 mbar. Once lyophilized, EFV SLNs were

completely characterized.

3.4 Determination of particle size, PDI and zeta potential of the EFV-SLNs

Mean diameter of the main population and polydispersity index (PDI) as a measure of the particle

size distribution was assessed using Zetasizer Nano ZS 90 (Malvern Instruments, UK). EFV SLN

formulations were diluted with double distilled water to weaken opalescence before particle size

analysis (Manjunath and Venkateswarlu, 2005). All measurements were carried out in triplicate.

The surface charge was determined by measuring the zeta potential of EFV SLN. Zeta potential

measurements were carried out at 25ᵒC with electric field strength of 23 Vcm-1.

5
3.5 Determination of entrapment efficiency (EE %) and loading efficiency (LE %) of the

EFV SLNs

EE (%) and LE (%) were calculated by determining the amount of nonencapsulated EFV in the

aqueous surfactant solution (Kalam, 2010). The EFV SLN dispersion was centrifuged (Remi

Instruments Ltd., Mumbai, India) at 16,000 rpm for 10 min at 4ᵒC. The concentration of EFV in

the aqueous phase was determined using UV–visible spectrophotometer (UV 1800, Shimadzu,

Japan) at ʎmax 247 nm. Values of EE % and LE % were calculated using Eqs. (1) and (2)

(Chalikwar et al., 2012)

% EE = X100 .. equation 1

−
% = × . .

Where,

Wa = amount of drug added to formulation

Ws = amount of free drug

WL= weight of lipid

3.6 Differential scanning calorimetry (DSC)

All measurements were carried out on an Indium calibrated DSC Q20 V24.9Build 121 (TA

instruments, USA) provided with refrigerated cooling system. Data was analysed using universal

analysis 2000 software (TA instruments, USA). Samples were weighed (2-3 mg) directly on DSC

aluminium pan and scanned between 25-150°C at a heating rate of 10°C min-1 in an inert

atmosphere of dry nitrogen (50 ml min-1). Sample pans were crimped with lids. An empty pan

with lid, was used as reference. The degree of crystallinity (% Crystallinity index, CI) was

calculated using the following equation (Teeranachaideekul, 2008):

Crystallinity Index (%CI) = 100 ..equation 3

6
Where, ΔH SLN and ΔH bulk material are the melting enthalpy (J g-1) of SLN dispersion and

bulk lipid, respectively.

3.7 X-ray powder diffraction (XRPD)

X-ray powder diffraction (XRPD) patterns of EFV, blank SLNs and EFV SLNs were recorded at

room temperature (Philips, Xpert MPD, Holland) with Cu target X-Ray tube (1.54Å), at 2 kW,

40 mA passing through nickel filter. Analysis was performed in a continuous mode with a step

size of 0.02° and step time of 1 sec over an angular range of 0-45° 2θ with Xe-filled counterate

or proportional detector.

3.8 In vitro drug release of EFV SLNs

In vitro drug release study of EFV SLN was performed using dialysis bag method (Dixit et al,

2010) [12]. Weighed quantity of EFV SLNs (equivalent to 100 mg of EFV) was dispersed in

phosphate buffer saline (PBS) pH 6.8 was filled in dialysis bag (Dialysis membrane 70, Himedia;

MWCO 12,000-14,000 daltons; pore size: 2.4 nm). The dialysis bag was closed from both sides

and placed in 250 ml PBS pH 6.8. 3 ml sample was withdrawn at regular interval for 24h and was

replaced by equal amount of fresh PBS (pH 6.8). The samples were analysed by UV Visible

spectrophotometer at 247 nm. The study was carried out in triplicate.

3.9 Bioanalytical method development for the estimation of EFV in lymph and plasma

EFV concentration was determined using Kromasil 100, C18 (5μm, 250×4.6 mm) column at 247

nm. The mobile phase consisted of a 68:32 (v/v) of acetonitrile: 10 mM Dipotassium hydrogen

phosphate buffer and the flow rate was 1.0 ml min-1. To the 50 µl of lymph/plasma sample (s), 1

ml of ethyl acetate was added and vortexed for 2 min. The organic phase was separated by

centrifugation at 4000 rpm and 4°C for 7 min. and supernatant was collected. 100 µl supernatant

was taken into 1.5 ml eppendorf tube and evaporated under nitrogen gas. The residue was

reconstituted using mobile phase. The method was adapted for analysis of EFV in lymph.

Calibration curves for EFV in lymph and plasma was prepared. The method was validated for

7
accuracy, precision, inter-day and intra-day variations, freeze thaw stability and bench top

stability.

3.10 Estimating lymphatic uptake of EFV SLN using chylomicron flow blocking approach

and biodistribution study

Male, Sprague Dawley rats weighing 250±20 g were used for intestinal lymphatic transport

studies. Animals were housed in propylene cages (38cm×23cm×10 cm) under laboratory

conditions of controlled environment of temperature 25±5ᵒC and 60±5% RH. Four rats per cage

were fed ad libitum with rodent’s food and allowing free access to drinking water. Animals were

fasted overnight. All surgical and experimental procedures were reviewed and approved by the

Institutional Animal and Ethics Committee (PERD/IAEC/2013/006).

Four groups of male Sprague Dawley rats, with each group containing 6 animals were subjected

to single oral dose. The 1st group was given saline solution, the 2nd group was given EFV-SLN,

3rd group was administered EFV-SLN after treatment of Cycloheximide. Cycloheximide is a

chylomicron blocker which inhibits the entry of drug into the lymphatic system. The animals were

treated with intra-peritoneal injection of cycloheximide (3 mg kg-1) dissolved in saline (3 mg ml-

1
, w/v). Fourth (4th) group was administered suspension of EFV in milli-Q water. The

formulations were administered orally. Approximately, 0.25 ml of blood samples was collected

at zero hour (pre-dose) followed by sample collection at 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 32h,

48h post-dose, from all animals of the each group. Samples were collected from jugular vein

cannula in heparinized collection vials. Plasma was separated by centrifugation at 4000 RPM for

7 min at 4°C. The plasma was then separated and stored at -80 °C till analysis.

For studying organ distribution profile, the vital organs i.e. brain, spleen, liver, kidneys and

lungs were excised, isolated and washed with deionised water. The organ samples were

homogenized in PBS pH 7.4 followed by extraction, deproteinization, drying and

8
reconstitution of the drug extract of the organs in 100 µL of mobile phase and accumulation

of drug in each organ was estimated using HPLC technique as discussed in section 3.9

3.11 Estimating lymphatic uptake of EFV SLN using direct method- mesenteric lymph duct

cannulated rat model

For intestinal lymphatic transport studies, Spague Dawley rats (350-400 gm) were selected.

Animals were fasted overnight. All surgical and experimental procedures were reviewed and

approved by the Institutional Animal and Ethics Committee. The surgical procedures were

undertaken as reported by Boyd et al. (2004). One hour preoperatively, the rat was given 1 ml of

olive oil by oral gavage to facilitate the identification of the mesenteric lymphatic duct. Olive oil

was administered before the surgery in order to facilitate the identification and cannulation of the

thoracic lymph duct. A fatty meal oral administration before surgery could not influence the

content of drug found in the lymph (Holm et al., 2002; Khoo et al., 2003).

Rats were anaesthetized using isoflurane with oxygen. Small incision was made in the abdomen;

mesenteric lymphatic duct was traced and cannulised. EFV SLN formulation (dose 38 mg kg-1)

was administered orally. Lymph was collected at the end of 4h and 6h, in heparinised tubes. EFV

concentration was determined using validated HPLC method.

3.10 Storage stability study of EFV SLNs

EFV SLNs were evaluated for their storage stability at different temperature conditions.

Lyophilized powder of EFV SLNs was sealed and stored in air tight glass bottles at 5 ± 2°C in

refrigerator and 30 ± 2°C/60 ± 5% RH, for a period of 2 months. Particle size, PDI, Zeta potential

and entrapment efficiency were determined at predetermined intervals (0, 7, 15, 30 and 60 days).

4. Results and Discussions:

One of the important parameter to be considered in the formulation of SLN is to obtain high drug

loading. Since the drug loading capacity of SLN depend on the solubility behavior of the drug in

the lipid melt (Westesen, 2000), the solubility of EFV in selected lipids and surfactants was

9
carried out for the screening. Based on the solubility study, it was found that Gellucire 44/14

(monoglycerides) and Compritol 888 ATO (triglycerides) were required in minimum quantity to

solubilise EFV as compared to other lipids evaluated. The chemical nature of the lipid is also

important because lipids that form highly crystalline particles with a perfect lattice (e.g.,

monoacid triglycerides) lead to drug expulsion. Lipids that are mixtures of mono-, di-, and

triglycerides and lipids containing fatty acids of different chain lengths form less perfect crystals

with many imperfections, offering space to accommodate the drugs (Vivek et al., 2007). Also,

studies have shown that the type of absorption pathway and subsequent transportation of drug is

significantly influenced by the two types of lipids viz. medium chain triglycerides and long chain

triglycerides. The long chain triglycerides are likely to augment the lymphatic transport of a

lipophilic drug substance leading to enhance oral bioavailability (Bandyopadhyay et al., 2012).

Hence, in the current study a mixture of monoglyceride (GEL) and triglyceride (COM) was

selected. Lipoid S 75 and Poloxamer 188 were selected as surfactant and cosurfactant

respectively, for the formulation development.

Homogenization followed by ultrasonication is a reliable, simple and reproducible method for

preparing SLN. Chloroform was found to be effective in homogenously dispersing EFV in the

lipid phase. Homogenization of the lipid phase with hot aqueous poloxamer solution for 15 min

at 15,000 rpm was sufficient to produce a coarse emulsion (with particle size of 250 nm and PDI

of 0.31). Further increase, in homogenization time and speed has however, not shown any

significant reduction in the particle size as well as PDI. Sonication time of the coarse emulsion

for 3 min. resulted in particle size 168.92±31.16 nm with a PDI of 0.219±0.060 (Table 1).

However, further increase in sonication time to 10 min has shown increase in particle size (upto

250 nm) as well as PDI (upto 0.42). This can be attributed to the agglomeration of nanoparticles

formed (Kumar et al., 2007; Manjunath and Venkateswarlu, 2005; Mehnert and Muller, 2001).

The optimized EFV SLN composition and optimized process variables are enlisted in Table 1.

10
To obtain stable EFV SLN with minimum size and narrow distribution, different percentages of

Lipoid S 75 and Poloxamer 188 and their effect on particle size and zeta potential was measured.

A mean particle size around 150 nm was obtained by reducing lipid percentage and increasing

surfactant concentration. However, the reduction in particle size was followed by decrease of drug

payload per ml and EE. Therefore, high concentration of surfactant was avoided to prevent

decrease in the EE and also due to toxic effects associated with surfactants. The formulation

having the highest encapsulation parameters, 85% (Table 1), was selected, which may be due to

its high lipophilicity (log P = 5.4) (Avachat and Parpani, 2014). This may be for the reason that

the long chain fatty acids attached to the glycerides results in increased accommodation of

lipophilic drugs (Venishetty et al., 2012). The mean particle size of the optimized SLN

formulation was found to be 168.92±31.16 nm with a PDI of 0.219±0.060 (Table 1). The LE of

the EFV SLN was found to be 39.4 % (Table 1).

Since the aqueous SLN dispersion is liable to undergo physical and chemical instability,

lyophilization seems to be the promising approach to increase the stability of SLN for extended

period of time. Furthermore, transformation into solid form offers principal possibilities of

incorporating SLN into pellets, tablets and capsules (Mehnert and Mader, 2001) In this regard, a

cryoprotectant is necessary to ensure ease of redispersion without any aggregation.

Cryoprotectants interact with the polar head groups of the surfactant and serve as a kind of

‘pseudo hydration shell’ (Mobley and Schreier, 1994). In this study, the mannitol (5% w/v) was

selected as cryoprotectant.

For the assessment of the drug–lipid interactions and the crystallinity of lipid matrices, DSC and

X-ray diffraction were carried out for the EFV, bulk lipids and surfactants, the physical mixture

of EFV and lipid in 1:1 ratio and the EFV SLNs, depicted in Fig 1 and Fig 2. DSC is widely used

to investigate the status of the lipid in SLN formulations and uses the fact that different lipid

modifications possess different melting points and enthalpies. The onset and melting

11
temperatures, melting enthalpy and the crystallinity index was calculated (Table 2). The

prominent endothermic peak at 139.88°C with melting enthalpy 35.67 J g-1 corresponding to the

melting temperature of EFV indicates the crystalline nature of EFV. Absence of an endothermic

peak in the physical mixture of drug with all excipients might be attributed to solubilization of

drug in lipid melt, before its own melting point (Fig 1). The absence of an endothermic peak

within the melting range of EFV in EFV SLN formulation unravels the solubilization of drug in

lipid matrix resulting in conversion of native crystalline state of the EFV to amorphous state.

Such behaviour of drug from crystalline form to amorphous form had been reported earlier for

ritonavir dispersion in gelucire as carrier (Sinha et al., 2010). The degree of crystallinity was

calculated from the enthalpies of EFV SLN and bulk lipid and is depicted in Table 2. The lower

crystallinity index (9.64%) for the lyophilized EFV SLN formulation compared to physical

mixture (15.45%) confers the embedment of EFV in the lipid matrix.

In order to investigate transformations and change in crystallinity, which may occurs during

preparation of the solid lipid nanoparticles and the influence of carrier in the possible phase

transformation, X-ray diffraction was performed for, a) EFV, b) blank SLNs and c) EFV SLNs,

shown in Fig 2. Both blank SLN and EFV SLNs peak pattern look similar in terms of peaks

positioning (Fig 2). Sharp and intense crystalline peaks at 2θ of 9.66° and 32.44° were observed

in the diffraction spectrum of EFV. However, broad peaks with reduced intensity of EFV were

observed in EFV SLNs. The decrease in crystallinity confirms an enhanced solubility of EFV in

lipid mixture. This indicates that the EFV was not in crystalline form in SLN (Figure.2). XRD

data complements the DSC data and clearly indicate the possible change in the crystallinity of

EFV after incorporation and formulation as SLN. The change in crystallinity of lipid and drug

also influence the release of drug from SLNs (Venishetty et al., 2012).

In vitro EFV release from EFV SLN was found to be prolonged in PBS pH 6.8. Cumulative EFV

released was 87.50% in PBS during 24h as shown in Fig 3. EFV SLN formulation exhibits a

12
typical biphasic pattern with initial burst release followed by sustained release (Fig 3). The 50%

EFV was released within 5h followed by controlled release of EFV over a period of 24h. The

initial in vitro burst release was probably caused by the EFV adsorbed on the nanoparticle surface

or precipitated from the superficial lipid matrix. The low solubility of the EFV in aqueous phase

could be the reason for the slow release of the EFV from the lipid matrices after initial burst

release. The subsequent sustained release of EFV could be probably due to diffusion of EFV

from the lipid matrix. The high lipid concentration prolongs the EFV release from SLN, attributed

to the good solubility of EFV in lipids (COM and GEL) and the homogenous distribution of

EFV within the lipid matrix (Burra et al., 2013; Chalikwar et al., 2012).

Following the drug release from dosage form and later absorption, a lipophilic molecule enter

into the enterocyte, it can reach the systemic blood circulation via two separate pathways, the

portal blood and the lymphatic transport. Potentially, drugs absorbed via the intestinal lymph

seems to gain their access into the lymphatic system via., three ways: the paracellular route with

the aid of absorption enhancers; the M cells and gut-associated lymphoid tissues (GALT); and a

transcellular route in association with the triglyceride core of the chylomicrons (O’Driscoll 2002).

Although the precise mechanisms of lymphatic transport have yet to be fully elucidated, the third

route is historically thought to be the major mechanism of lymphatic delivery of lipophilic drugs

when formulated with lipid-based vehicles (O’Driscoll 2002). It has been proved that the

incorporation of the lipophilic molecule into the chylomicron inside the enterocyte is an essential

step in the cascade of the lymphatic absorption process of lipophilic drugs (Dahan et al., 2005;

Gershkovich et al., 2007; Trevaskis et al, 2008). Thus, to block the chylomicron flow with

blockers provides a novel tool to investigate the fraction of drug transported by the intestinal

lymphatic system. Besides, this method has been proved to be not affecting non-lymphatic

absorption pathways and no adverse effects were found (Dahan et al., 2005). Cyclohexamide is

one of the chylomicron flow blocking agent. It has been found that cycloheximide can block the

13
lymphatic transport through two mechanisms, viz., the blockage of chylomicron flow and the

inhibition on the phagocytic activity of M cells and further block the pathway of lymphatic

transport via the M cells.

Hence, in this study, we hypothesized that drug absorption from the intestinal tract to the systemic

circulation include two pathways: the portal blood and the lymphatic transport. When the

lymphatic absorption component is blocked by a chylomicron flow blocker (cyclohexamide), the

portal blood pathway is the only route of drug absorption. In compliance, the amount of EFV,

from the chylomicron flow inhibiting approach were compared with the mesenteric lymph duct

cannulated rat model and drug accumulation in spleen, major lymphatic organ, and liver.

Plasma and lymph samples obtained from different treated groups were analysed and

concentrations at different time points was calculated using validated HPLC method (linearity:

50-1000 ng ml-1 ; precision: RSD <15%; and accuracy: 96.6-105.8%). Pharmacokinetic

parameters for EFV SLN, EFV SLN + Cycloheximide and EFV are listed in Table 3. Figure 4

and Table 3 shows plasma concentration versus time curves, which clearly shows that area under

the curve for EFV SLNs treated group is less as compared to Cycloheximide + EFV SLN treated

groups, analysed after administration of equivalent doses. This indicates that when the

chylomicron production is not blocked, a significant part of EFV is diverted to the lymphatics.

While on blocking the chlyomicron production, the portal blood route is the only pathway for

EFV absorption, resulting in higher EFV plasma concentration in Cycloheximide + EFV SLN

treated group.

In EFV SLN treated (without cyclohexamide) group, a significant part of administered drug might

have been uptaken by lymphatic system through chylomicron formation, resulting in lower AUC

in plasma (Table 3). This was confirmed by the estimation of concentration of EFV in lymph

samples, via mesenteric lymphatic duct cannulation. The lymph samples were analyzed at two

different time points (4h and 6h), in the EFV SLN treated groups and API treated group of rats.

14
Amount of EFV in lymph at 4h and 6h was estimated to be 98.73 ng ml-1 and 147.11 ng ml-1,

respectively. Thus, it can be concluded that significant amount of administered EFV was taken

up by lymphatic system. Lymphatic uptake of EFV SLNs was further supported by the presence

of significant amount of EFV in spleen (Table 3). In addition, EFV concentration in different

tissues such as brain, liver, and kidney was also estimated. It was found that EFV reaches to liver,

however, not to brain and kidney after 48h of drug administration. The amount of EFV reaching

to liver is higher when administered as an EFV in comparison to EFV SLN, both in

cyclohexamide treated group as well as saline treated group. This confirms that EFV is taken up

by portal system and is available to undergo first pass metabolism in liver. While in case of EFV

SLN (without cyclohexamide) treated group, drug from EFV SLN reaching to liver is 44.70%

less in comparison to EFV treated group (Table 3), indicating avoidance of first pass metabolism.

In contrast, in cyclohexamide treated EFV SLN group, the EFV reaching to liver is comparatively

higher than EFV SLN (without cyclohexamide) group. This may be due to blocking of lymphatic

transport pathway by cyclohexamide, as a result of which maximum amount of EFV is taken up

by portal system via systemic circulation. This results in higher amount of EFV reaching to liver

and thus higher probability of undergoing first pass metabolism. These findings are supported by

an earlier study of Alex et al (2011). Alex et al, reported that the SLNs can preferably be absorbed

by peyer’s patches and transported to intestinal lymphatic, bypassing the liver, thereby, enhancing

the oral bioavailability of the drug (Alex et al, 2011).

Thus, the results from lymphatic transport study and tissue distribution indicates that a

considerable part of the EFV by-passed the systemic circulation and was recovered in the diverted

lymph via chylomicron uptake mechanism, when administered as EFV SLN. This may be

attributed to the small size of the EFV SLN as well as the presence of long chain triglycerides in

the EFV SLN composition.

15
Accelerated stability studies were conducted on optimized EFV SLN using the particle size, PDI

zeta potential and EE (%) as the prime parameters. There was a slight increase in the particle size

during the three months storage from the 160 ± 5.30 nm to 183.0 ± 5.15 nm with insignificant

change in PDI (i.e. initially which was 0.235 ± 0.18 and after 2 months it was 0.290 ± 0.03) and

zeta potential varied from -36.0 to -34.0. The EE (%) of the optimized batch initially was found

to be 85.6 ± 9.72% while that, after 2 months was found to be 83.89 ± 0.14% indicating that the

drug can retain within the nanoparticles for the sufficient period of time. On storage of the EFV

SLN, there were no significant alterations in the size, PDI zeta potential and EE % of the SLNs

(Table 4). Hence, they were found to be stable at 25 ± 2 ◦C/60 ± 5% RH for a total period of 3

months.

5. Conclusion:

The targeting of the ARV drug to the HIV virus in oral lymphatic region is the prime focus of the

ARV therapy regimen nowadays. Among various lipid delivery system, SLN serve as promising

candidate for transport of lipophilic drugs, like NNRTIs, through the lymphatic region via

chylomicron uptake. EFV SLNs were prepared by hot homogenization technique followed by

ultrasonication. In vivo pharmacokinetic parameters revealed that long chain triglyceride

containing EFV SLN showed enhanced bioavailability of the EFV along with targeted drug

delivery of EFV to the reservoir sites of HIV virus, i.e lymphatics. Higher availability of the EFV

in lymphatics will ensure that significant parts of the administered dose will by-pass the portal

circulation to reach systemic circulation. This will ensure reduced first pass metabolism of the

drug and higher bioavailability of the EFV. In addition, by-passing portal system, will also open

up the desired possibility of ARV drug co-administration with Rifampicin. Eventually, the

prolonged release of drug at reservoir site will help in reducing the dosing frequency along with

better patient compliance and disease management.

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Conflict of interest: The authors report none conflict of interest.

Acknowledgements: The author gratefully acknowledges Mylan Pharmaceuticals Ltd

(Hyderabad), for the gift samples of drug. This work was funded by the Ministry of Chemicals

and Fertilizers, Government of India to NIER-G for the MS Pharm. work of VM, RJ and KP.

Authors are thankful to PERD Centre, Ahmedabad for providing the usage of facilities and

Director, NIPER-G for the research funding.

References:
Alex, M.R.A., Chacko, A.J., Jose, S., Souto, E.B., 2011. Lopinavir loaded solid lipid

nanoparticles (SLN) for intestinal lymphatic targeting. Eur. J. Pharm. Sci. 42, 11–18.

Avachat, A.M., Parpani, S.S., 2014. Formulation and Development of Bicontinuous

Nanostructured Liquid crystalline particles of Efavirenz, Colloid. Surf. B Biointerface.

http://dx.doi.org/10.1016/j.colsurfb.2014.12.014.

Bandyopadhyay, S., Katare, O.P., Singh, B., 2012. Optimized self nano-emulsifying systems of

ezetimibe with enhanced bioavailability potential using long chain and medium chain

triglycerides. Colloid. Surf. B Biointerface. 100, 50– 61.

Boyd, M., Risovic, V., Jull, P., Choo, E., Wasan, K.M., 2004. A stepwise surgical procedure to

investigate the lymphatic transport of lipid-based oral drug formulations: Cannulation of the

mesenteric and thoracic lymph ducts within the rat. J. Pharmacol. Toxicol. Method. 49, 115-

120.

Burra, M., Jukanti, R., Janga, K.Y., Sunkavalli, S., Velpula, A., Ampati, S., Jayaveera, K.N.,

2013. Enhanced intestinal absorption and bioavailability of raloxifene hydrochloride via

lyophilized solid lipid nanoparticles. Adv. Powder Technol. 24, 393–402.

17
Chalikwar, S.S., Belgamwar, V.S., Talele, V.R., Surana, S.J., Patil, M.U., 2012. Formulation and

evaluation of Nimodipine-loaded solid lipid nanoparticles delivered via lymphatic transport

system. Colloid. Surf. B Biointerface. 97, 109– 116.

Dahan, A., Hoffman, A., 2005. Evaluation of a chylomicron flow blocking approach to investigate

the intestinal lymphatic transport of lipophilic drugs. Eur. J. Pharm. Sci. 24 (4), 381-388.

Dane, K.Y., Nembrini, C., Tomei, A.A., Eby, J.K., O'Neil, C.O., Velluto, D., Swartz, M.A.,

Inverardi, L., Hubbell, J.A., 2011. Nano-sized drug-loaded micelles deliver payload to lymph

node immune cells and prolong allograft survival. J. Control. Rel. 156, 154–160.

Dixit, A.R., Rajput, S.J., Patel, S.G., 2010. Preparation and Bioavailability Assessment of

SMEDDS Containing Valsartan. AAPS PharmSciTech. 11(1), 314-321.

Gershkovich, P., Shtainer, D., Hoffman, A., 2007. The effect of a high-fat meal on the

pharmacodynamics of a model lipophilic compound that binds extensively to triglyceride-rich

lipoproteins. Int. J. Pharm. 333, 1–4.

Gupta, U., Jain, N.K., 2010. Non-polymeric nano-carriers in HIV/AIDS drug delivery and

targeting, Adv. Drug Deliv. Rev. 62, 478–490.

Holm, R., Porter, C.J.H., Mullertz, A., Kristensen, H.G., Charman, W.N., 2002. Structured
triglycerides vehicles for oral delivery of halofantrine: examination of intestinal lymphatic

transport and bioavailability in conscious rats. Pharm. Res. 19, 1354–1361.

Hu, L., Tang, X., Cui, F., 2004. Solid lipid nanoparticles (SLNs) to improve oral bioavailability
of poorly soluble drugs. J. Pharm. Pharmacol. 56, 1527–1535.
Kalam, M.A., Sultana, Y., Ali, A., Aqil, M., Mishra, A.K., Chuttani, K., 2010. Preparation,

characterization, and evaluation of gatifloxacin loaded solid lipid nanoparticles as colloidal

ocular drug delivery system. J. Drug Target. 18, 191–204.

Khoo, S.M., Shackleford, D.M., Porter, C.J.H., Edwards, G.A., Charman, W.N., 2003. Intestinal

lymphatic transport of halofantrine occurs after oral administration of a unit-dose lipid-based

formulation to fasted dogs. Pharm. Res. 20,1460–1465.

18
Kumar, V.V., Chandrasekar, D., Ramakrishna, S., Kishan, V., Rao, Y.M., Diwan, P.V., 2007.

Development and evaluation of nitrendipine loaded solid lipid nanoparticles: influence of wax

and glyceride lipids on plasma pharmacokinetics. Int. J. Pharm. 335, 167–175.

Li, X., Lin, X., Zheng, L., Yu, L., Lv, F., Zhang, Q., Liu, W., 2008. Effect of poly(ethylene

glycol) stearate on the phase behaviour of monocaprate / Tween80 / water system and

characterization of poly (ethylene glycol) stearate-modified solid lipid nanoparticles. Colloid.

Surf. A 317, 352-359.

Manjunath, K., Venkateswarlu, V., 2005. Pharmacokinetics, tissue distribution and

bioavailability of clozapine solid lipid nanoparticles after intravenous and intraduodenal

administration. J. Control Rel. 107, 215–228.

Mehnert, W., Mader, K., 2001. Solid lipid nanoparticles production, characterization and

applications, Adv. Drug Deliv. Rev. 47, 165–196.

Mobley, W.C., Schreier, H., 1994. Phase transition temperature reduction and glass

transformation in dehydroprotected lyophilized liposomes, J. Control. Rel. 31, 73–87.

Negi, J.S., Chattopadhyay, P., Sharma, A.K., Rama, V., 2013, Development of solid lipid

nanoparticles (SLNs) of lopinavir using hot self nano-emulsification (SNE) technique.

Eur. J. Pharm.Sci. 48, 231–239.

O’Driscoll, C.M., 2002. Lipid-based formulations for intestinal lymphatic delivery. Eur. J. Pharm.

Sci. 15, 405–415.

Renekin, E.M., Wiig, H., 1994. Limits to steady state lymph flow rates derived from plasma-

to-tissue uptake measurements. Microvascular Res. 47, 318-328.

Sinha, S., Ali, M., Baboota, S., Ahuja, A., Kumar, A., Ali, J., 2010. Solid Dispersion as an

Approach for Bioavailability Enhancement of Poorly Water-Soluble Drug Ritonavir. AAPS

PharmSciTech. 11(2), 518–527.

19
Teeranachaideekul, V., Souto, E.B., Müller, R.H., Junyaprasert, V.B., 2008. Physicochemical

characterization and in vitro release studies of ascorbylpalmitate-loaded semi-solid

nanostructured lipid carriers (NLC gels). J. Microencapsul. 25, 111–120.

Trevaskis, N.L., Charman, W.N., Porter, C.J., 2008. Lipid-based delivery systems and intestinal

lymphatic drug transport: a mechanistic update. Adv. Drug Deliv. Rev. 60, 702–716.

Venishetty, V.K., Chede, R., Komuravelli, R., Adepu, L., Sistla, R., Diwan, P.V., 2012. Design

and evaluation of polymer coated carvedilol loaded solid lipid nanoparticles to improve the

oral bioavailability: A novel strategy to avoid intraduodenal administration. Colloid. Surf. B

Biointerface. 95,1– 9.

Vivek, K., Reddy, H., Murthy, R.S.R., 2007. Investigations of the effect of the lipid matrix on

drug entrapment, in vitro release, and physical stability of olanzapine-loaded solid lipid

nanoparticles. AAPS PharmSciTech. 8 (4), Article 83.

Westesen, K., 2000. Particles with Modified Physico-Chemical Properties, their Preparation and

Uses, US Patent No. 6197349.

Table 1: Composition, optimised process variables and characterization parameters of optimized

EFV SLNs
Composition (%) Optimised process variable Characterization

Efavirenz High speed 15 min Particle size=160.12±5.20

homogenizer time nm

Gelucire 44/14 High speed 15000 rpm PDI= 0.225±0.025

(30%) homogenizer speed

Compritol 888 Sonication time 3 min Zeta potential=-36.0

ATO (20%)
Lipoid S 75 Sonication impulse 5 sec on & 5 sec EE(%)=85.6%; LE %=39.4
off %
(25 %)
Poloxamer 188
(5%)

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 Table 2: Melting peak, enthalpy and crystallinity index of EFV SLN and their respective bulk and
physical mixtures.
Melting Point (ᵒC) Enthalpy CI (%)
Bulk Lipid 70.25, 44.05 204.8 100
Physical Mixture 43.11 94.94 30.90
SLN 71.70 29.62 9.64
CI indicate crystallinity index.

Table 3 Pharmacokinetic parameters (Cmax, Tmax and AUC) and Tissue distribution of EFV in rats

when treated with EFV SLN, EFV SLN + cyclohexamide and pure drug, EFV (n=6)

Group Cmax(µg ml-1) Tmax(h) AUC Liver Spleen Brain

(µg min-1 ml-1) (ng ml -1) (ng ml -1) (ng ml -1)
EFV-SLN 253.17 2.0 1758.69 77.00 263.30 ND
treated
Cycloheximide+ 312.97 2.0 3118.92 130.00 117.45 ND
SLN treated
Pure EFV 204.93 1.0 1612.59 172.30 112.00 ND

Table 4: Effect of storage at 5 °C and 30±2° C/60±5 %RH on the particle size, PDI and %EE of
EFV SLN for 2 months
Stability 5 °C 30±2° C/60±5 %RH
parameter 0 Month 1 Month 2 Month 0 Month 1 Month 2 Month

Particle 160.12±5.20 178.05±4.17 183.00±5.15 160.12±5.20 169.42±6.72 178.36±8.56

size (nm)
PDI 0.225±0.025 0.282±0.022 0.290±0.030 0.225±0.025 0.274±0.096 0.284±0.045
Zeta -36.0 -34.0 -34.0 -36.0 -34.4 -34.1
potential
(mV)
%EE 85.6% 84.05% 83.89% 85.6% 84.63% 84.40%

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