Optimization of nanostructured lipid carriers for Zidovudine delivery using a microwave-assisted production method

Accepted Manuscript Optimization of nanostructured lipid carriers for Zidovudine delivery using a microwave-assisted production method

S.M.T. Cavalcanti, C. Nunes, S.A.C. Lima, J.L. Soares-Sobrinho, S. Reis PII: DOI: Reference:

S0928-0987(18)30273-2 doi:10.1016/j.ejps.2018.06.017 PHASCI 4559

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

21 March 2018 28 May 2018 18 June 2018

Please cite this article as: S.M.T. Cavalcanti, C. Nunes, S.A.C. Lima, J.L. SoaresSobrinho, S. Reis , Optimization of nanostructured lipid carriers for Zidovudine delivery using a microwave-assisted production method. Phasci (2018), doi:10.1016/ j.ejps.2018.06.017

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ACCEPTED MANUSCRIPT Optimization of nanostructured lipid carriers for Zidovudine delivery using a microwave-assisted production method

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S. M. T. Cavalcantia,b, C. Nunesa*, S. A. C. Limaa, J. L. Soares-Sobrinhob, S. Reisa

LAQV, REQUIMTE, Department of Chemistry, Faculty of Pharmacy, University of

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Porto, Portugal.

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Rua de Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal.

Core of Medicine and Correlated Quality Control – NCQMC, Department of

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Pharmaceutical Sciences, Federal University of Pernambuco, Brazil.

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Rua Arthur de Sá, s/n, Cidade Universitária, 50, 740-521 Recife, PE, Brazil.

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* Corresponding author at:

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Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Fax: +351 226093483.

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E-mail address: [email protected].

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ACCEPTED MANUSCRIPT Abstract An adapted methodology for obtaining lipid nanoparticles that only uses the microwave reactor in the synthesis process was developed. The method has the following features: one-pot, one-step, fast, practical, economical, safe, readiness of scaling-up, lack of organic solvents and production of nanoparticles with low polydispersity index (PDI)

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(below 0.3).

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This new method was applied for the development of nanostructured lipid carriers

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(NLC) loaded with a hydrophilic drug, the antiretroviral agent zidovudine (AZT). The aim of the present work was to develop, evaluate and compare optimized NLC

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formulations produced by two different methods – hot ultrasonication and microwaveassisted method. The development and optimization of the NLC formulations were

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supported by a Quality by Design (QbD) approach.

All formulations were physicochemically characterized by the same parameters. The

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optimized formulations presented a suitable profile for oral administration (particle size

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between 100 to 300 nm, PDI < 0.3 and negative zeta potential > -20 mV). Furthermore, the morphologies assessed by TEM showed the spherical shape and confirmed the

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results obtained by DLS. Both formulations were physically stable for at least 45 days, non-toxic on Jurkat T cells and drug release studies showed a controlled release of AZT

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under gastric and plasma-simulated conditions.

Keywords Lipid nanoparticles; Quality by Design (QbD); design of experiments (DOE); microwave; anti-viral drug; in vitro drug release assays; Jurkat cells.

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ACCEPTED MANUSCRIPT 1.

Introduction

Lipid nanoparticles were first developed in the early 1990s. Today, they are among the most attractive nanoparticles for drug delivery, due to the combination of some characteristics, such as particle size, low toxicity, physical stability, controlled release properties, high drug loading and excellent tolerability (Chakraborty et al., 2009;

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Moghimi et al., 2005; Severino et al., 2012). Lipid nanoparticles are prepared as

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colloidal arrangements with a solid lipid matrix held together by a surfactant. Its

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constituents are excipients recognized as safe and known to be biocompatible; therefore, these nanoparticles are Generally Recognized as Safe (GRAS) (Muchow et al., 2008;

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Severino et al., 2012). A variety of techniques has been developed for lipid nanoparticle production. High-pressure homogenization and microemulsion are the main techniques

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used to prepare lipid nanoparticles encapsulating different types of drugs. However, these methods remain with particular drawbacks such as high energy inputs, inefficient

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thermal heating in homogenization-based techniques (Shah et al., 2015), and the risk of

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metal contamination when ultrasounds is used in microemulsion techniques (Betts et al., 2013).

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For these reasons, different production procedures for lipid nanoparticles have been widely investigated in recent years (last decade). In fact, Shah et al. in previous works,

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have used microwave energy to replace thermal heat in the heating step of the constituents to produce solid lipid nanoparticles (SLNs), which highlighted several advantages for this purpose (Shah et al., 2017; Shah et al., 2014). The replacement of the thermal heating process by a temperature-controlled microwave heating process, produced SLNs with smaller particle sizes, narrow polydispersity, higher encapsulation efficiency and loading capacity, and increased physical stability (Shah et al., 2014). However, the process involves a second production step that requires the immediate

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ACCEPTED MANUSCRIPT dispersion of the fresh made formulation into cold water in a ratio of 1:50, which profoundly decreases the yield of the process and makes it time consuming. This is the main difference of our methodology. When the microwave process finishes, the lipid nanoparticles are ready; there is no need of dispersing them in water. This step is time consuming and dilutes a lot the suspension. The increased amount of water decreases

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the number of particles by mL, which for an industrial process is not recommended, as

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it can increase the number of production steps to remove the excess of water. Thereby in

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this work, we present a one-pot and one-step methodology for obtaining lipid nanoparticles that only uses the microwave reactor. This technique includes some

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advantages, such as, it is cost-effective, safe, and reproducible; it offers the possibility of easily being scaled-up; the lack of organic solvents; and, the production of small

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particles diameter with a polydispersity index (PDI) usually below 0.3. Once we recognize the importance of the Quality by Design (QbD) approach in the development

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of products and processes aligned with Risk Management, identification and control of

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critical control points (ICH, 2005, 2009),we have use them in this study. The encapsulated drug, zidovudine (AZT), is an nucleoside reverse transcriptase

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inhibitor (NRTI) applied as a first antiretroviral agent and most widely used of human immunodeficiency virus (HIV), alone or associated with other anti-viral drugs. AZT is a

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class I drug by the Biopharmaceutical Classification System (BCS) (high solubility and high permeability) (Lindenberg et al., 2004) having water solubility of 25 mg.mL-1 at 20 ºC (IARC, 2000) and log P near zero (Thomas and Panchagnula, 2003). Despite its therapeutic efficacy, AZT presents several drawbacks, like: low oral bioavailability (Klecker Rw Jr Fau - Collins et al., 1987), dose-dependent side effects bone marrow toxicity resulting in granulocytopenia and anemia (Chow and Hamburger, 1991; Lutton et al., 1990; Richman et al., 1987). Additionally, the drugs used on anti-retroviral

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ACCEPTED MANUSCRIPT therapy cannot reach in efficient concentrations the central nervous system (CNS), which is an important reservoir of the HIV (Chakraborty et al., 2009; Phillips and Tsoukas, 1992). Moreover, AZT plasma concentration occurs immediately after its administration (Lobenberg and Kreuter, 1996), which leads to an increase in the frequency and dosage of the treatment regimen, due to the requirement of an uniform

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systemic level of the drug throughout the course of the HIV therapy. Thereby, the

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encapsulation of AZT in lipid-based nanoparticles may improve the lymphatic uptake,

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avoid hepatic first pass metabolism and its controlled delivery may reduce the dose requirement and avoid dose dependent toxicity (Chakraborty et al., 2009; Phillips and

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Tsoukas, 1992).

The aim of the present work was to evaluate and compare the optimum experimental

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conditions for the development of nanostructured lipid carriers (NLC) by two different microemulsion methods – conventional hot ultrasonication (Ferreira et al., 2015) and

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one-step microwave technique, both as potential antiretroviral delivery systems for

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AZT. A preliminary study was performed to select some critical parameters and fix others. In the whole study, a design of experiments (DOE) was applied. Namely, a

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factorial design, central composite design and response surface to select the better formulation to each production method. The key answers, throughout the study, were

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particle size, PDI and loading capacity. The optimized developed formulations were characterized regarding morphology, in vitro drug release, storage stability and their effect on Jurkat T cells viability.

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Materials and methods 2.1. Materials

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ACCEPTED MANUSCRIPT All chemicals were at least analytical grade and used as received without further purification. Zidovudine was provided from Ítaca® (batch HVZ0210304). The solid lipid, Precirol® ATO 5, was provided by Gattefossé (Nanterre, France) and the liquid lipid, Miglyol® 812, was acquired from Acofarma (Madrid, Spain). Polysorbate 80 (Tween®80) was supplied by Merck (Darmstadt, Germany). Ultrapure water was

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obtained by reverse osmosis process with a conductivity inferior to 0.1 μS cm-1 (Milli-

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Q, Sartorius Arium® pro, Sartorius Weighing Technology; Gettingen, Germany). Jurkat

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T (human lymphoma) cell line was obtained from ATCC (Philadelphia, PA, United States of America (USA) and fetal bovine serum (FBS), penicillin-streptomycin

Methods

2.2.1. Preliminary screening

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2.2.

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antibiotics mixture and RPMI 164 were purchased from Gibco® (Paisley, UK).

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Before the experimental design was constructed, two parameters were fixed: the used

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surfactant, Tween®80, once it is a steric surfactant with low toxicity; and the aqueous phase, double deionized water at pH 5.5, once at this pH it is ensured the predominance

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of the neutral form of AZT (pKa 9.68 (IARC, 2000)), facilitating its permanence in the lipid phase. Pre-formulation studies were performed with different solid and liquid

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lipids at different ratios between the lipid phase (LP) and the aqueous phase (AP) (1:20 to 1:50 w/w). The selection was based on the criteria that the lipid mixture should enhance the solubility of AZT and the tested lipids were: Precirol® ATO 5, Compritol® ATO 888, Gelucire®, Cetyl palmitate, Oleic acid and Miglyol®.

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ACCEPTED MANUSCRIPT 2.2.2. Experimental design and optimization The QdD approach of this study aimed to design optimized NLC-AZT and M-NLCAZT (Figure S1 -supporting information) formulations and their manufacturing processes by hot ultrasonication and a novel microwave-assisted technique, respectively. Ishikawa diagrams were applied to identify and select the critical

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parameters involved in each production methodology (Figure 1). The quantitative

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critical independent variables selected to the experimental design of NLC-AZT were

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sonication time and amounts of Tween 80® and solid lipid. For M-NLC-AZT the critical independent variables were the amounts of Tween 80® and solid lipid. The lower (-1)

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and higher (+1) levels of each selected critical parameters were chosen based on the pre-formulation studies and literature research. All other variables were set at fixed

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levels.

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ACCEPTED MANUSCRIPT Figure 1. Ishikawa diagram of critical parameters of both hot ultrasonication and microwaveassisted techniques. * This parameters is only applied to the microwave technique.

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The experimental design methodology was the full factorial or central composite design, with replication of center points (level 0). Four axial were applied (± α, where α

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= 1.4142 or √2) to evaluate the effects of independent variables and their interactions on

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dependent variables. Experiments were randomly performed and response surface (RS) analysis was applied to identify the conditions that provided optimized NLC-AZT and

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M-NLC-AZT formulations.

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The chosen dependent variables of the experiment were mean particle size (constraints: 100 to 300 nm), PDI (constraints: minimize up to 0.3) and loading capacity (constraints: maximize).

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The Statistica 8.0 (Statsoft®, Inc.) software was employed to make the matrix design, statistical analysis, graphs, mathematical models and RS construction. Statistical

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analysis was performed using ANOVA at 95 % confidence level (p-values < 0.05).

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2.2.3. Preparation of NLC and M-NLC NLC and M-NLC were prepared by two different microemulsion techniques: conventional hot ultrasonication method and a one-step microwave-assisted method, respectively.

2.2.3.1. Hot ultrasonication method The lipid phase constituted by solid lipid Precirol®ATO 5, the liquid lipid Miglyol® 812, the stabilizer Tween® 80 and optionally AZT, were melted at 80 ºC, a temperature

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ACCEPTED MANUSCRIPT above the phase transition temperature of Precirol® ATO 5. The melted lipid phase was then dispersed in pre-warmed ultrapure water also at 80 ºC and sonicated for 2 to 20 min with a frequency amplitude of 50%, 130 KHz (model VCX130 with CV-18, Sonics & Materials, Newtown, CT, USA). It was then cooled down to room temperature (RT), allowing the inner oil phase to solidify establishing NLCs dispersion in the aqueous

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phase.

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2.2.3.2. One-step microwave-assisted method

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The microwave-assisted method can be described by being a one-pot and one-step

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production process. Briefly, all the constituents, Precirol® ATO 5, Miglyol® 812, Tween® 80, ultrapure water an, optionally AZT were placed in borosilicate glass vessels

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(35 mL) and heated at 90 ºC for 10 min with constant stirring (about 900 rpm) in a microwave reactor (CEM Discover SP®, 2.45 GHz, 0-300 W, ActiVentTM using

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Synergy™ software). After, it was quickly cooled to 70 ºC by the microwave reactor

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system, under a stream of N2. Finally, the formulations were cooled at RT.

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2.2.4. Mean particle size and polydispersity index (PDI) measurements Particle size analysis was performed by dynamic light scattering (DLS), using a particle size analyzer (Brookhaven Instruments, Holtsville, NY, USA). Prior to the measurements, all samples were diluted (1:200) using ultrapure water to yield a suitable scattering intensity (Kcps 300-500). DLS data was analyzed at 25°C with a fixed light incidence angle of 90°. The mean hydrodynamic diameter (Z-average) and the PDI were determined as a measure of the width of the particle size distribution. The size and PDI of the analyzed samples were obtained by calculating the average of ten runs.

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ACCEPTED MANUSCRIPT 2.2.5. Zeta potential measurements The zeta potential was determined by measuring of the electrophoretic mobility of the nanoparticles using a zeta potential analyzer (Brookhaven Instruments, Holtsville, NY, USA). Samples were diluted (1:200) with ultrapure water and were analyzed at 25 °C. The zeta potential (Zeta) of the sample was obtained by calculating the average of six

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runs (each one with ten cycles).

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2.2.6. Morphology determination

The morphology of optimized NLC-AZT and M-NLC-AZT was assessed by

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Transmission Electron Microscopy (TEM), by placing 10 µL of prediluted (100x) NLC suspensions on a copper-mesh grid and left to rest for 2 min at RT. After this period, the

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excess was removed with filter paper and to enhance contrast, 10 µL of 1% (w/v) uranyl acetate solution were placed on the grid and left to rest for 5 sec. The excess was

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removed with filter paper. The samples were observed in a JEM-1400 Transmission

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Electron Microscope (JEOL Ltd., Tokyo, Japan) with an acceleration voltage of 80 kV.

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2.2.7. Loading capacity (LC) and entrapment efficiency (EE) The amount of AZT present in the formulations was determined using an indirect

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method. Lipid nanoparticles suspensions were diluted 200x and filtrated with Amicon® Ultra Centrifugal Filters Ultracell-50 kDa (EMD Millipore, Darmstadt, Germany) at 3400 xg for 15 min, at 18ºC using a Heraeus™ Multifuge™ X1R centrifuge (Thermo Scientific, Waltham, MA, USA). The resulting supernatant was collected, for AZT quantification at 266 nm wavelength (maximum AZT absorbance peak) in a V-660 UV/Vis Spectrophotometer (Jasco Inc., Easton, MD, USA). A calibration curve of AZT in ultrapure water was used to determine its concentration.

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ACCEPTED MANUSCRIPT Loading capacity (LC) and entrapment efficiency (EE) are expressed as a percentage. LC is the amount of AZT encapsulated compared to the total amount of lipid, whilst EE refers to the amount of AZT encapsulated compared to the total amount of drug added. The loading capacity (LC) was calculated as follows:

surfactants

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The entrapment efficiency (EE) was calculated as follows:

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LC (%) = (total amount of drug – unentrapped drug) x 100 / total amount of lipids and

In vitro drug release measurements

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2.2.8.

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EE (%) = (total amount of drug – unentrapped drug) x 100 / total amount of drug

In vitro release studies were performed using a cellulose dialysis bag (Float-a-

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Lyzer®G2, SpectrumLaboratories, Inc., CA, USA Germany) diffusion technique filled with 1.5 mL of the optimized formulations. In order to mimic the particles path in the

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body after oral administration, samples were placed at 37 °C under 300 rpm stirring

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(IKA®-Werke RT15-P Hot Stirring Plate; Germany). To simulate the transit from stomach to intestine, samples were placed first for 4 h in 76 mL of simulated gastric

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fluid (HCl solution, pH 1.2) and then placed in 76 mL of a buffered solution containing potassium dihydrogen phosphate, pH 7.4, as described in the United States

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Pharmacopeia, USP-NP 26, until the end of the experience (P.). At regular intervals of time, aliquots of 1.5 mL were withdrawn and the same volume of fresh buffer was replaced to maintain sink conditions. The AZT release was quantified by UV/Vis spectroscopy, using calibrations curves obtained with the different buffers. The results are expressed as the mean values of two independent assays. In order to assess the main mechanisms responsible for the AZT release, mathematical models were fitted to the experimental data, namely zero order, first order, Higuchi, Peppas–Korsmeyer and

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ACCEPTED MANUSCRIPT Hixon–Crowell (Barzegar-Jalali et al., 2008). Regression coefficient (r2) was calculated to determine the best-fit model.

2.2.9. Stability studies In order to evaluate the stability of optimized NLC-AZTs formulations, a short-term

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stability were mean particle size, PDI, zeta potential and LC.

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storage study was carried out at 4 ºC for a period of 45 days. The indicators of storage

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2.2.10. Cytotoxicity Studies

Jurkat T cells were grown in RPMI 1640 (GIBCO, Paisley, UK) supplemented with

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10% heat-inactivated fetal bovine serum and 1% penicillin–streptomycin, at 37°C in a

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humidified atmosphere containing 5% CO2. Jurkat T cells were plated on 96-well plates at 105 cells per well and were treated with NLCs and NLC-AZTs up to 2.0 mg.mL-1 in

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lipid equivalent to 15 µM in AZT for 24 hours. Treated and control cells (untreated and

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0.2% (v/v) Triton X-100) viability was assessed by the methylthiazoletetrazolium (MTT) assay. In brief, 10 µL of a 5 mg.mL-1 solution of MTT was added to the wells and incubated at 37 ºC for 3 h. The reaction was stopped by using 100 µL of 10% (w/v)

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SDS for 16 h. Cell growth was evaluated by measuring the absorbance at 545 nm, using

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a microplate spectrophotometer (Synergy™ HT, Biotek, USA). Results were expressed as the percentage of the metabolic activity of treated cells relative to untreated cells, and all experiments were performed in quadruplicate. GraphPad Prism software (version 6, GraphPad Software, USA) was used for statistical analysis of the results using student (unpaired) t-test and one-way ANOVA test. All the other results were presented as mean and standard deviation (SD). The differences were assumed statistical significant when p < 0.05 (95% confidence level).

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ACCEPTED MANUSCRIPT 3.

Results and Discussion 3.1. Preliminary screening

Among the tested lipids, the solid lipid Precirol® ATO 5 and the liquid lipid Miglyol® – 812 were selected, because their mixture provided the best solubilization of AZT. The

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ratio between LP:AP of 1:40 w/w was selected for the conventional method. For the microwave-assisted method, this ratio was not satisfactory to the answers size and PDI.

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In this case, the ratio LP:AP of 1:50 w/w was selected. Additionally, in the microwave-

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assisted method other parameters such as production time and cooling temperatures were evaluated. The selected parameters were: 10 min at 90 ºC for the nanoparticles

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production and fast cooling to 70 ºC in the microwave system. The stirring rate, size of

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the magnetic bar, selected microwave vessel (35 mL) and total volume of formulation were fixed based on previous “know-how” acquired during the development of this methodology.

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The amount of AZT was different for each production technique. In the conventional method the drug is placed in the lipid phase and heated after, thus it was selected an amount of drug saturation in that lipid phase (2 mg or 0.7% w/w of lipid phase).

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Whereas the microwave-assisted method is a one-pot reaction so, all the components are

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placed in the same vessel, at the same time. In this case it was selected a greater amount of drug (15% w/w of lipid phase) to be possible to increase the LC.

3.2. Experimental design, optimization and validation Studies were carried out to establish the conditions that provided a maximum LC, minimum PDI and nanoparticles size between 100 to 300 nm in all NLC-AZTs formulations.

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ACCEPTED MANUSCRIPT 3.2.1. NLC production by a hot ultrasonication method First, a full factorial design 23 was built to enable the evaluation of three quantitative critical factors and their interactions at the lower and higher levels selected: sonication time (2 and 20 min); quantity of Tween® 80 (T-80) (25 and 100 mg) and quantity of solid lipid (SL) (200 and 270 mg). Eight randomized experiments were performed. It is

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noteworthy that the quantity of total lipids was kept constant at 300 mg throughout the

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study.

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The individual interactions effects of the factors for each answer were analyzed by Pareto charts (Figure 2). Through the Pareto chart and multivariate analysis it was

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possible to realize that the sonication time and quantity of T-80 were the most significant factors. Both have a negative impact on LC, which indicates that the best LC

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results should be obtained when these factors are adjusted to their lower level (-1) (Figure S2a). The individual effect of quantity of SL was also considered significant,

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indicating the higher level (+1).

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For the answer size, only the factor quantity of T-80 was significant, with a negative impact, as indicated for the LC answer (Figure 2b). For PDI answer (Figure 2c), all

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factors were statistically significant. For these reasons, only the LC answer was selected to be optimized and the quantity of SL factor was set at 250 mg and consequently 50 mg

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of liquid lipid, an intermediate value between the high and low levels previously evaluated. Thus, a central composite design was employed (Table S1), resulting in 13 experiments (with axial points and replication of central point) for analysis. Thus, the influence and significance of the two most critical factors and their interactions for the LC answer (which are quantity of T-80, in the range from 9.5 to 115.5 mg and sonication time, in the range from 30 sec to 24 min) were assessed. The data was

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ACCEPTED MANUSCRIPT analyzed using ANOVA. RS charts were built to identify the optimal conditions for

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each dependent variable (Figure 3).

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ACCEPTED MANUSCRIPT Figure 2. Pareto chart of the standardized effects for the answers LC (a), Size (b) and PDI (c) for 23 full factorial design of NLC-AZT formulation study. The length of each bar is proportional to the absolute value of the associated regression coefficient or estimated effect. The effects of all parameters and interactions were standardized (each effect was divided by its standard error). The order in which the bars are displayed corresponds to the order of

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importance of the effect. When the vertical line crosses an effect bar in the chart, it indicates

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that this effect is statistically significant with 95% reliability (p = 0.05).

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Figure 3. Response surface chart for the answer LC of NLC-AZT experimental design.

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A statistically significant quadratic model, accounting for 84 % of the variance (r2 = 0.84), was fitted to the data. This model describes the correlation between observed and predicted answers values. The quadratic regression model is given by equation S1 (see supporting information). From the analysis of the RS methodology and its desirability function, 2 min and 20 sec of sonication time and 31 mg of quantity of T-80 were selected. With these values, it is predicted that an optimum LC value for the developed formulation NLC-AZT is obtained. Size and PDI answers are also satisfactory for these selected conditions.

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ACCEPTED MANUSCRIPT The experimental value of LC answer for the optimized formulation was considered to

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be in good agreement with the predicted by the models (Figure 4).

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Figure 4. Loading capacity percentages obtained by the mathematical models and experimental

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conditions. No statistical significant differences were observed (p > 0.05).

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3.2.2. NLC production by a one-step microwave-assisted method

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Some parameters were previously fixed due to the results of the pre-formulation studies, which made possible to select two quantitative critical factors and their lower (−1) and

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upper (+1) levels: quantity of SL, in the range from 63.3 to 86.7 mg, and quantity of T80, from 79.3 to 220.7 mg. The central composite design was performed, resulting in 13

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experiments (with axial points and replication of central point), to analyze the influence and significance of selected factors and built the RS. Randomized experiments were performed, according to the matrix design presented in Table S2, considering different combination of the factors for each experiment. It is noteworthy to mention that the total quantity of lipids was kept at 100 mg throughout the study. For the answers LC and size, all factors (quadratic and linear) and their interactions were statistically significant; while for PDI answer only the quadratic factors were

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ACCEPTED MANUSCRIPT statistically significant, in 95% of confidence . The results can be seen in Figure 5 through the Pareto chart and multivariate analysis with calculated p-values = 0.05. ANOVA was used to analyze the relevance of the models for each answer. By fitting the experimental data was possible to plot the RS to LC, Size and PDI answers and thus identify the best conditions in the range evaluated in this study (Figure 6).

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A statistically significant quadratic model (r2), accounting for 95% and 93% of the

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variance for answers LC and PDI respectively, was fitted to the data. These models describe the correlation between observed and predicted answers values. The quadratic

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model of size answer was not considered because r2 < 0.8. Thereby, the RS

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methodology was only used as an indicative for the optimization of this answer. The quadratic regression model obtained is described by equations S2 and S3 (see

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supporting information).

The critical values, obtained from the analysis of the RS methodology and its

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desirability function, for the optimized formulations were fulfilled: 158 mg T-80 and 73

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mg SL for answer LC and 150 mg T-80 and 75 mg SL for answer PDI. Size answer was

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also satisfactory for these selected conditions.

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Figure 5. Pareto chart of the standardized effects for the answers LC (a), Size (b) and PDI (c) for 23 full factorial design of M-NLC-AZT formulation study.

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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT Figure 6. Response surface chart for the answers: LC (a), Size (b) and PDI (c) of M-NLC-AZT formulation study.

From the analysis of the RS methodology, it was possible to determine the process conditions to, simultaneously, optimize all the answers of interest in this study, since the

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critical values for each answer were similar. This way, the quantity of T-80 was fixed in 158 mg and the quantity of SL in 75 mg (and 25 mg of liquid lipid) for the M-NLC-

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AZT optimized formulation.

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The experimental values of LC and size answers of the M-NLC-AZT optimized

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predicted values of models (Figure 7).

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formulation were analyzed and were considered to be in good agreement with the

Figure 7. Validation of the experimental values of selected M-NLC-AZT formulation with its PDI and

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predicted results.

LC (%). No statistically significant differences were observed

(p > 0.05).

3.3. Physico-chemical characterization of the optimized formulations Whitish translucent and low density NLC suspensions were obtained by both methods. The formulations were characterized in terms of mean particle size, PDI, zeta potential, LC and entrapment efficiency (EE) (Table 1). All these properties may influence the

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ACCEPTED MANUSCRIPT formulation´s stability and also influence their future interactions with cells and tissues (Sperling and Parak, 2010; Sussman et al., 2008).

Size (nm)

PDI

Zeta (mV)

LC (%)

EE (%)

NLC-P*

307 ± 2

0.206 ± 0.02

-34.7 ± 0.3

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NLC-AZT

266 ± 4

0.168 ± 0.01

-29 ± 2

0.31 ± 0.04

44 ± 3

M-NLC-P*

85 ± 2

0.260 ± 0.01

-15 ± 2

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M-NLC-AZT

113 ± 3

0.216 ± 0.01

-20 ± 1

1.41 ± 0.02

22 ± 2

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Formulation

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Table 1. Size, PDI, -potential, drug LC and EE of optimized NLC formulations.

Mean ± standard deviation (SD) (n = 3). * Placebo formulations.

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The average diameter of the formulations is in the nanometric range, suitable for oral,

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dermal and gastrointestinal absorption (des Rieux et al., 2007). It is described in literature that above 200 nm, particles are more readily incorporated by the

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macrophages, which are, together with the lymphocytes, target cells for the proposed

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approach (Martins et al., 2012; Schöler et al., 2002). In contrast, particles with mean size below 200 nm are potentially more long-time circulating than larger particles and particles smaller than 20–30 nm (renal excretion) (Gaumet et al., 2008; Moghimi et al.,

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2001). Generally, nanoparticles with mean size between 50 and 250 nm are considered

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to be promising drug delivery systems since they have the potential to improve therapy due of their capability to overcome biological barriers (Alexis et al., 2008). For the optimized NLC formulations the PDI values range between 0.168 ± 0.01 and 0.260 ± 0.01 (Table 1). The PDI values below 0.3 are indicative of monodispersed and uniform diameters of nanoparticles populations (Lopalco et al., 2015). Zeta potential is a key factor in the evaluation of the physical stability of colloidal dispersions, since it is a function of the surface charge that reflects the electrostatic repulsive interactions between particles (Komatsu et al., 1995). Zeta potential values of

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ACCEPTED MANUSCRIPT all formulations are depicted in Table 1. In general, particle aggregation or flocculation is prevented when zeta potential is > |30| mV. However, |20| mV is also considered an accepted value (Mishra et al., 2009; Müller et al., 2001; Souto et al., 2004), especially when the particles have a steric stabilization agent, as in these cases with T-80 (Mitri et al., 2011). Due to the electrostatic repulsion between NPs and the cellular membrane,

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negatively charged particles can reduce cellular uptake; however, they show less

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cytotoxicity than cationic nanoparticles, usually associated with cell membrane

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disruption and consequent cell death (Ekambaram et al., 2012).

M-NLC-AZT formulation, delivered a LC value approximately 5 times greater than the

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NLC-AZT. Both formulations present low EE, which is probably related with the high initial amount of AZT used in development of these formulations.. The priority was

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given to optimize the response LC in the experimental designs. The different production processes and the ratio between constituents affected all the

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studied properties of NLC. Thus, regarding the two presented microemulsion methods,

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one can be more adequate than the other, depending on the drug's characteristics, route

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of administration, and design of the formulation´s development.

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ACCEPTED MANUSCRIPT 3.4. In vitro AZT release assay The solubility of a drug in the lipid matrix becomes a very important controlling factor for the drug´s release from NLC (Pinto et al., 2014), since the drug is incorporated in the lipid matrix of the nanoparticles system either in dissolved or dispersed form (Müller et al., 2002). The in vitro AZT release profile from the free drug, NLC-AZT

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and M-NLC-AZT were investigated in two conditions designed to simulate

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physiological and gastric environments. The results show 52% release of free AZT in

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gastric medium and 100% for the physiological environment in 4 h (data not shown). A release of AZT of about 40%, in the gastric medium was verified for both

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formulations. In physiological environment, 100% release was achieved at 45 h for NLC-AZT and 28 h for M-NLC-AZT formulations (Figure 8). The NLC formulations`

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exhibited a burst release during the early hours of study in physiological environment (about 50% in 5 h).

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The release results were analyzed using mathematic models for drug release kinetics.

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The AZT release from the NLC, for both methods and conditions studied, was best fitted to the Higuchi model (R2>0.98, Figure S4). Thereby, the release of AZT from

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both formulations is driven by controlled diffusion. The slope of each linearization corresponds to the release rate constant (KH). The KH values are very similar between

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the two media (pH 1.2 and 7.4), which means that AZT release is not pH dependent. At pH 7.4, it is possible to see that the release of AZT from both formulations, follows two regimes: one until 10 h and another from that time on. Although the second regime is also controlled by diffusion, this is a much slower process, which possibly corresponds to the AZT release that is more near the core of NLC.

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Figure 8. In vitro AZT release profile NLC-AZT ( ) and M-NLC-AZT (

), (a) simulating

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gastric environment and body temperature (pH 1.2 at 37 ° C), and (b) simulating physiological

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environment and body temperature (pH 7.4 at 37 °C). The data are the mean and SD for n = 2.

3.5. Morphology

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The images obtained by TEM of the optimized NLC formulations, Figure 9, revealed a spherical shape and allowed to verify the results obtained by DLS. A slight reduction in

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particle size was observed compared to DLS data. This difference was expected and is

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related with the fact that DLS measures the hydrodynamic diameter (Mehnert and Mäder, 2001). Additionally, the DLS measurements were performed in aqueous media,

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where the lipid particles are highly hydrated; while during the preparation of samples for TEM, both the water surface as the water present within the nanoparticle matrix are

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removed by evaporation, which leads to shrinkage of the particles, and thus determining a slightly size lower (Dubes et al., 2003).

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Figure 9. TEM images of NPs formulations: (A) NLC-AZT, amplification: 25,000 x; (B) M-

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NLC-AZT, amplification: 100,000 x.

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3.6. Stability studies

The physical stability of the optimized NLC formulations, regarding size, PDI and zeta

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potential, show that the formulations were able to maintain their properties up to 45

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days when stored as aqueous suspensions at 4 °C and protected from light (Figure 10). Thus, the both formulations maintained the size between 100 to 300 nm, PDI of up to

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0.3 and negative zeta potential > -20 mV. No change was found to be significant in any

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formulation for the same period.

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Figure 10. Comparative storage stability of NLC-AZT and M-NLC-AZT formulations over a period of 45 days. The bars represent the size of the nanoparticles (left Y axis) and markers

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stand for zeta potential (right Y axis) from the day of preparation to 45 days. The values

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3.7. Cytotoxicity studies

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represent the mean of 3 independent assays and respective SD.

The Jurkat T cells viability was studied in the presence of NLC-AZT, M-NLC-AZT and

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free AZT. NLC placebo prepared by the two studied methodologies, at a concentration of 2 mg.mL-1, did not decrease Jurkat T cells viability (Figure 11). Also, exposure of

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Jurkat T cells to therapeutic concentrations of AZT (1 to 10 M) (Francke et al., 2000) as free solution or incorporated within NLC (NLC-AZT and M-NLC-AZT) did not induce any significant effect on cell viability. It has been reported that cellular toxicity of AZT requires 10 to 15-fold higher concentrations, as observed previously in HeLa, MT4 and human T cells (Chiu and Duesberg, 1995; Liotard et al., 2006). Both methodologies produced NLC-AZTs formulations biocompatible with the target cells of AZT in the context of HIV therapy.

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Figure 11. Jurkat T cells viability. In vitro cytotoxicity of free AZT, NLC-AZT and M-NLC-

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AZT and respective placebos, upon 24 h incubation. Data expressed as mean ± SD (n=3).

Conclusions

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An innovative, one-step approach for the development of NLC by a microwave-assisted

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method is reported in this work. Although, microwave energy has been previously used in the production of lipid nanoparticles, this is, to the best of our knowledge, the first

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work that reports its application for the development of NLC. Furthermore, this modified method is less time consuming and allows the minimum operator influence,

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which delivers reproducible results for every synthesis, and enables the scale-up. The use of a one-pot and single step represents the added value of this new methodology. It was confirmed that the replacement of the conventional hot homogenization process followed by ultrasonication, with a single temperature controlled microwave heating process produces NLC with smaller particle sizes; equivalent PDI; higher loading capacity; alike physical stability, biocompatibility on Jurkart T lymphocytes, and controlled release of the studied drug. Additionally, all the processes were optimized

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ACCEPTED MANUSCRIPT according to a QbD methodology that maximized the characteristics of both formulations. In order to validate completely this new method for the development of drug delivery systems based on lipid nanoparticles, other formulations with different components and drugs should be tested. Regarding the AZT delivery, both the optimized formulations are suitable for its oral administration and are considered safe,

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which makes them potential carriers for AZT delivery.

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Acknowledgements

This work received financial support from the European Union (FEDER funds

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POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership

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Agreement PT2020 UID/QUI/50006/2013.

The authors are also grateful to Dr Rui Fernandes (Histology and Electron Microscopy

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Service – Instituto de Investigação e Inovação em Saúde, Universidade do Porto) for the

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expertise and technical assistance with transmission electron microscopy. SCL thanks Operação NORTE-01-0145-FEDER-000011. CN thanks FCT for the

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investigator grant IF/00293/2015. Cavalcanti and Soares-Sobrinho thank the Facepe

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(1316-4.03/12) and CNPq (482954/2013-2) for financial support.

Declarations of interest: none

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ACCEPTED MANUSCRIPT Figure captions

Figure 1. Ishikawa diagram of critical parameters of both hot ultrasonication and microwave-assisted techniques.

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* This parameters is only applied to the microwave technique.

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Figure 2. Pareto chart of the standardized effects for the answers LC (a), Size (b) and PDI (c) for 23 full factorial design of NLC-AZT formulation study. The length of each bar is

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proportional to the absolute value of the associated regression coefficient or estimated effect.

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The effects of all parameters and interactions were standardized (each effect was divided by its standard error). The order in which the bars are displayed corresponds to the order of

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importance of the effect. When the vertical line crosses an effect bar in the chart, it indicates that this effect is statistically significant with 95% reliability (p = 0.05).

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Figure 3. Response surface chart for the answer LC of NLC-AZT experimental design.

Figure 4. Loading capacity percentages obtained by the mathematical models and experimental

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conditions. No statistical significant differences were observed (p > 0.05).

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Figure 5. Pareto chart of the standardized effects for the answers LC (a), Size (b) and PDI (c) for 23 full factorial design of M-NLC-AZT formulation study.

Figure 6. Response surface chart for the answers: LC (a), Size (b) and PDI (c) of M-NLC-AZT formulation study.

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ACCEPTED MANUSCRIPT Figure 7. Validation of the experimental values of selected M-NLC-AZT formulation with its predicted results.

PDI and

LC (%). No statistically significant differences were observed

(p > 0.05).

Figure 8. In vitro AZT release profile NLC-AZT ( ) and M-NLC-AZT (

), (a) simulating

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gastric environment and body temperature (pH 1.2 at 37 ° C), and (b) simulating physiological

RI

environment and body temperature (pH 7.4 at 37 °C). The data are the mean and SD for n = 2.

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Figure 9. TEM images of NPs formulations: (A) NLC-AZT, amplification: 25,000 x; (B) M-

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NLC-AZT, amplification: 100,000 x.

Figure 10. Comparative storage stability of NLC-AZT and M-NLC-AZT formulations over a

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period of 45 days. The bars represent the size of the nanoparticles (left Y axis) and markers stand for zeta potential (right Y axis) from the day of preparation to 45 days. The values

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represent the mean of 3 independent assays and respective SD.

Figure 11. Jurkat T cells viability. In vitro cytotoxicity of free AZT, NLC-AZT and M-NLC-

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AZT and respective placebos, upon 24 h incubation. Data expressed as mean ± SD (n=3).

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Graphical abstract

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