development and characterization of lipid nanoparticles ... · transporte de fármacos, absorção...

December 2014

Development and Characterization of Lipid

Nanoparticles prepared by Miniemulsion Technique

Clara Patrícia Andrade Lopes

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisor: Professor Luís Joaquim Pina da Fonseca

Examination Committee

Chairperson: Professor Duarte Miguel de França Teixeira dos Prazeres

Supervisor: Professor Luís Joaquim Pina da Fonseca

Member of the committee: Doctor Dragana Popovic Correia de Barros

i

Acknowledgments

I would like to start by thanking my supervisor, Professor Luis Fonseca who accepted me

and provides me all the means to developed this research thesis, sharing his knowledge with

me and guiding me through this project with optimism and patience (even through the rough

times and the dangerously close deadlines) and for always being present when needed.

I would like to express my sincere gratitude to all BEBL members, professors and

colleagues, for all opinions, questions, answers and suggestions to improve my project. To all

my lab partners, for making me feel so welcome, giving me all the scientific and moral support

and especially for contributing with the good environment which in my opinion is truly important

because provides a greater motivation and commitment of the people.

I would like to acknowledge all my friends (leirienses, escuteiros and fculianos) that in all

my life, in one way or another, have helped and support me with their truly friendship.

Finally, a deep acknowledgment goes to my family, my mother and sisters, who never

stopped to support me through all my academic years, always encouraging my success and

putting up with my bad mood and bad retorts on the endless hours of work. And last, but

certainly not the least, to my father who taught me to work hard, ever smile and never give up!

ii

Abstract

Lipid nanoparticles are a promising alternative to traditional colloidal drug carriers. The

main reason for this is related with the well-known conception that lipids promote oral drug

absorption, because they undergo the same physiological mechanisms of food lipid digestion.

Lipid nanoparticles also offer unique properties such as small size, large surface area,

increased drug loading and stability (especially for lipophilic drugs), possibility of controlled drug

release, no toxicity and high bioavailability.

In the present work a medium chain fatty acid (MCFA) SLNs and NLCs based on MCFA

and a natural oil were successfully prepared by sonication technique and employing a nonionic

surfactant. The two anti-TB drugs, rifampicin and pyrazinamide, are entrapped into obtained

lipid nanoparticles. The developed particles were characterized in terms of particle size,

polydispersity index, zeta potential, morphology and encapsulation efficiency. Mean particle size

of all formulations ranged between 69 ± 5 nm to 601 ± 100 nm, showing a suitable size for oral

administration. The zeta potential obtained was negatively enough to ensure a good physical

stability of the particles. Morphological studies by TEM showed spherical to oval SLNs and RIF-

NLCs with well-defined periphery. The rifampicin encapsulation efficiency range between 67 ±

7% and 85 ± 5%, while pyrazinamide encapsulation efficiency range between 14 ± 8% to

29±15%. In conclusion, despite being essential several further studies to ensure the efficacy of

obtained lipid nanoparticles, the results from the present work pose a strong argument for lipid

nanoparticles as a promising strategy for the oral delivery of anti-TB drugs.

Keywords: Oral Administration; Solid Lipid Nanoparticles; Medium Chain Fatty Acid; Nonionic

Surfactant; Tuberculosis

iii

Resumo

As nanopartículas lipídicas são uma promissora alternativa aos sistemas coloidais de

transporte de fármacos, absorção oral de fármacos, dado que sofrem os mesmos mecanismos

fisiológicos da digestão dos lípidos provenientes dos alimentos. As nanopartículas lipídicas

também oferecem propriedades únicas como tamanho reduzido, grande área de superfície,

aumento da capacidade de carga, estabilidade e libertação controlada do agente activo dentro

da partícula (especialmente para fármacos lipofílicos), aumento da biodisponibilidade e

reduzida toxicidade.

No presente trabalho, nanopartículas lipídicas sólidas (SLN) de um ácido gordo de

cadeia média (MCFA) e vectores lipídicos nanoestruturados (NLC) baseados na combinação

desse MCFA e óleo natural foram preparados com sucesso através da técnica de sonicação e

recorrendo a um tensioactivo não iónico. Dois fármacos anti-TB, a rifampicina e a pirazinamida,

foram internalizados dentro das partículas lipídicas. Estas foram caracterizadas em termos de

dimensão, índice de polidispersão, potencial zeta, morfologia e eficiência de encapsulação. A

dimensão média de todas as formulações variou entre 69 ± 5 nm e 601 ± 100 nm, exibindo

dimensões apropriadas para administração oral. O potencial zeta obtido foi suficientemente

negativo para garantir uma boa estabilidade física das partículas. Os estudos de morfologia

feitos através de Microscopia Electrónica de Transmissão (TEM) mostram que tanto as SLNs

como as RIF-NLC apresentam uma forma esférica ou oval e uma periferia bem definida. A

eficiência de encapsulação da rifampicina variou entre 67 ± 7% e 85 ± 5%, enquanto a da

pirazinamida variou entre 14 ± 8% e 29±15%. Concluindo, apesar de ser necessário realizar

outros estudos, os resultados apresentados neste trabalho permitem prever que as

nanopartículas lipídicas obtidas são uma alternativa promissora para o transporte de fármacos

anti-TB por via oral.

Palavras-chave: Administração Oral; Nanopartículas Lipídicas Sólidas; Ácidos Gordos de

Cadeia Média; Tensioactivo Não-Iónico, Tuberculose

iv

Table of Contents

Acknowledgments .......................................................................................................................... i

Abstract .......................................................................................................................................... ii

Resumo ......................................................................................................................................... iii

Table of Contents .......................................................................................................................... iv

List of Figures ................................................................................................................................ vi

List of Tables ................................................................................................................................ vii

List of Abbreviations .................................................................................................................... viii

Motivation and Aims of the Thesis ................................................................................................ ix

1. Introduction ............................................................................................................................ 1

1.1. Lipid Nanoparticles ............................................................................................................ 2

1.1.1. Lipid Nanoparticles as Oral Drug Delivery System ....................................................... 5

1.1.1.1. Toxicological Concerns ........................................................................................... 11

1.1.2. Preparation Techniques for Lipid Nanoparticles ......................................................... 13

1.1.2.1. High Pressure Homogenization Technique ............................................................. 13

1.1.2.1.1. Hot Homogenization Technique .......................................................................... 14

1.1.2.1.2. Cold Homogenization Technique ........................................................................ 14

1.1.2.2. Microemulsion Technique ....................................................................................... 15

1.1.2.3. Ultrasonication Technique ....................................................................................... 15

1.1.2.4. Solvent Emulsification Evaporation Technique ....................................................... 16

1.1.2.5. Double Emulsion Technique ................................................................................... 16

1.1.2.6. Solvent Emulsification Diffusion Technique ............................................................ 17

1.1.2.7. Solvent Injection Technique .................................................................................... 17

1.1.2.8. Phase Inversion Temperature Technique ............................................................... 18

1.1.2.9. Microchannel/Microfluidic Technique ...................................................................... 18

1.1.3. Characterization of Lipid Nanoparticles....................................................................... 19

1.1.3.1. Measurement of Particle Size ................................................................................. 19

1.1.3.1.1. Dynamic Light Scattering..................................................................................... 19

1.1.3.2. Measurement of Zeta Potential ............................................................................... 20

1.1.3.3. Encapsulation Efficiency.......................................................................................... 22

1.1.3.4. Morphology .............................................................................................................. 22

1.1.3.4.1. Transmission Electron Microscopy ...................................................................... 23

1.1.3.5. Lipid Crystallinity ...................................................................................................... 23

1.2. Tuberculosis – Review .................................................................................................... 24

2. Materials and Methods ........................................................................................................ 28

2.1. Materials .......................................................................................................................... 28

2.1.1. Lipids ........................................................................................................................... 28

v

2.1.2. Surfactant .................................................................................................................... 28

2.1.2.1. Hexadecane ............................................................................................................ 29

2.1.3. Bioactive Compounds ................................................................................................. 29

2.1.3.1. Rifampicin ................................................................................................................ 29

2.1.3.2. Pyrazinamide ........................................................................................................... 30

2.1.3.3. β-carotene ............................................................................................................... 30

2.1.4. Water ........................................................................................................................... 31

2.2. Methods ........................................................................................................................... 31

2.2.1. Preparation of Lipid Nanoparticles .............................................................................. 31

2.2.1.1. Lyophilisation of Lipid Nanoparticles ....................................................................... 33

2.2.2. Characterization of Lipid Nanoparticles....................................................................... 33

2.2.2.1. Particle Size ............................................................................................................. 33

2.2.2.2. Zeta Potential .......................................................................................................... 33

2.2.2.3. Particle Morphology ................................................................................................. 34

2.2.2.4. Encapsulation Efficiency.......................................................................................... 34

3. Results and Discussion ....................................................................................................... 37

3.1. Study of Fabrication Parameters ..................................................................................... 37

3.1.1. Empty SLN .................................................................................................................. 37

3.1.1.1. Influence of Temperature ........................................................................................ 41

3.1.1.2. Influence of Lipid Content ........................................................................................ 41

3.1.1.3. Influence of Sonication ............................................................................................ 42

3.1.1.4. Lyophilisation ........................................................................................................... 43

3.1.1.5. Long-Term Stability ................................................................................................. 44

3.1.2. Empty NLC .................................................................................................................. 46

3.2. Bioactive Compounds Loaded SLNs and NLCs ............................................................. 47

3.2.1. β-carotene ................................................................................................................... 48

3.2.2. Rifampicin .................................................................................................................... 49

3.2.3. Pyrazinamide ............................................................................................................... 50

4. Conclusions and Further Works .......................................................................................... 52

References .................................................................................................................................. 54

vi

List of Figures

Figure 1 – Schematic structure of solid lipid nanoparticle (a) and nanostructured lipid carrier (b).

....................................................................................................................................................... 2

Figure 2 - Models of drug incorporation in Solid Lipid Nanoparticles. From [32]. ......................... 3

Figure 3 - Models of drug incorporation in Nanostructured Lipid Carriers. From [50]. .................. 4

Figure 4 - Various mechanisms of enhancement of drug bioavailability in the presence of lipids.

....................................................................................................................................................... 7

Figure 5 - Schematic diagram of lipid nanoparticles formation in the microchannel system. From

[113] ............................................................................................................................................. 18

Figure 6 -Correlation differences between small and large particles. From [126]. ..................... 20

Figure 7 - Zeta-potential of a nanoparticle in solution. From [126]. ............................................ 21

Figure 8 – Schematic structure of M. tuberculosis cell wall and action site of first-line anti-TB

drugs. Intercalation of hydrophilic arabinogalactan and hydrophobic mycolate containing layers

creates an extremely impermeable envelope for antibiotic penetration. Small molecules and

nutrients are transported through porin channels that are deposited through these layers.

Adapted from [140]. ..................................................................................................................... 26

Figure 9- Structural formula of hexadecane ................................................................................ 29

Figure 10- Structural formula of rifampicin .................................................................................. 29

Figure 11 - Structural formula of pyrazinamide ........................................................................... 30

Figure 12 – Structural formula of β-carotene .............................................................................. 30

Figure 13 – Lipid nanoparticles production. Left: Lipid phase and aqueous phase in the warm

water bath under same conditions (temperature and agitation). Top Right: Lipid phase (on the

right) and aqueous phase (on the left) after weight. Middle Right: Addition of hot aqueous phase

in melt lipid phase. Bottom Right: Sonication of hot pre-emulsion. ............................................. 32

Figure 14 – Pyrazinamide standard solutions used in constructing of the calibration curve, after

reacting with alkaline sodium nitroprusside. Pyrazinamide concentration increases from left to

right. ............................................................................................................................................. 35

Figure 15 - Rifampicin calibration curve ...................................................................................... 35

Figure 16 - β-carotene calibration curve ..................................................................................... 36

Figure 17 - Pyrazinamide calibration curve ................................................................................. 36

Figure 18- Z-ave and PDI of empty SLNs with different content of MCFA (SLN_1 represents the

lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from

a lowest temperature (a) to highest temperature (d). .................................................................. 39

Figure 19 - Zeta Potential of empty SLNs with different content of MCFA (SLN_1 represents the

lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from

a lowest temperature (a) to highest temperature (d). .................................................................. 40

vii

Figure 20 - Macroscopic aspect of empty SLNs: (left) - non-sonicated sample; (right) –

sonicated sample ........................................................................................................................ 42

Figure 21 – Powder resulting of SLN_1b_II lyophilisation .......................................................... 43

Figure 22 – Particle Size Distribution by Intensity of SLN_1b_II before and after lyophilisation. 44

Figure 23 - Particle Size Distribution by Intensity of SLN_4b_I after one day and 7 months after

production. ................................................................................................................................... 45

Figure 24 – TEM image of SLN_4b_I after 7 months. ................................................................ 46

Figure 25 – Differences in macroscopic appearance of empty and loaded NLCs immediately

before and after sonication. ......................................................................................................... 47

Figure 26- TEM image of β-carotene loaded NLC. ..................................................................... 48

Figure 27 - TEM image of rifampicin loaded NLC. ...................................................................... 50

Figure 28 - TEM image of pyrazinamide loaded NLC ................................................................. 51

List of Tables

Table 1 – Examples of different Drug-Loaded Lipid Nanoparticles and their potential benefits for

application in oral administration route. ......................................................................................... 8

Table 2 - Dosage forms of TB nanomedicine. From [149]. ......................................................... 27

Table 3 - Composition % (w/w) of bioactive compounds loaded to lipid nanoparticles. ............. 31

Table 4 - Physicochemical characteristics of empty SLN at different conditions (mean ± SD, n =

6). ................................................................................................................................................ 38

Table 5 – Physicochemical characteristics of the SLN_1b_II. Values are mean ± SD, n = 3 ..... 44

Table 6 - Physicochemical characteristics of the SLN_4b_I. Values are mean ± SD, n = 3. ..... 45

Table 7 – Physicochemical characteristics of the empty NLC. Values are mean ± SD, n = 3. ... 46

Table 8 - Physicochemical characteristics of the β-carotene lipid nanoparticles. Values are

mean ± SD, n = 3. ....................................................................................................................... 48

Table 9 - Physicochemical characteristics of the rifampicin lipid nanoparticles. Values are mean

± SD, n = 3. ................................................................................................................................. 49

Table 10 - Physicochemical characteristics of the pyrazinamide lipid nanoparticles. Values are

mean ± SD, n = 3. ....................................................................................................................... 51

viii

List of Abbreviations

ATM Atomic Force Microscopy RES Reticulo-Endothelial System

CMC Critical Micellar Concentration RIF Rifampicin

CNS Central Nervous System SD Standard Deviation

DLS Dynamic Light Scattering SEM Scanning Electron Microscopy

DSC Differential Scanning Calorimetry SLN Solid Lipid Nanoparticle

EE Encapsulation Efficiency STM Scanning Tunnelling Microscopy

ETB Ethambutol TB Tuberculosis

FDA Food and Drug Administration TEM Transmission Electron Microscopy

FFEM Freeze Fracture Electron Microscopy UV Ultraviolet

GI Gastrointestinal Vis Visible

GRAS Generally Recognized as Safe w/o Water-in Oil

HHT Hot Homogenization Technique w/o/w Water-in Oil-in Water

HLB Hydrophilic-Lipophilic Balance w/w Weight/Weight

HPH High Pressure Homogenization WHO World Health Organization

INH Isoniazid XDR Extremely Drug Resistant

KatG Catalase Peroxidase Z-Ave Z-Average

LC Loading Capacity ZP Zeta Potential

MCFA Medium Chain Fatty Acid

MDR Multi-Drug Resistant

MTT (4,5-imethylthiazol-2-yl)2,5-dyphenyl-

tetrazolium bromide

NLC Nanostructured Lipid Carrier

NP Nanoparticle

o/w Oil-in Water

OSPS Optical Single Particle Sizing

PCS Photon Correlation Spectroscopy

PDI Polydispersity Index

PIT Phase Inversion Temperature

PLG Poly(lactide-co-glycolide)

PYZ Pyrazinamide

R2 Determination Coefficient

ix

Motivation and Aims of the Thesis

Tuberculosis (TB) remains one of major health problems in the world. In 2012, there were

8.7 million new cases of TB globally, with 1.4 million deaths in the same year. Treatment of TB

is typically a 6-month regimen comprising an initial intensive phase of rifampicin (RIF), isoniazid

(INH), pyrazinamide (PYZ), and ethambutol (ETB) daily for 2-months, followed by a 4-month

continuation phase of RIF and INH given 3 times a week. The complexity of the treatment and

patient non-compliance result in an incomplete or inadequate treatment and subsequently in the

emergence of multi-drug-resistance. The epidemic of drug-resistant tuberculosis has spread

quickly in some areas due to the convergence of resistant strains of M. tuberculosis in high-risk

patients (e.g., those with human immunodeficiency virus/acquired immunodeficiency syndrome)

and high-risk environments (e.g., hospitals and prisons). Moreover, the administration of anti-

TB drugs for a long period of time can lead to the incidence of unpleasant side effects, which

decreases patient adherence and, therefore, increases costs of therapy. To overcome these

drawbacks, new oral anti-TB formulations are required.

Apart from some particular situations, the oral route is the first choice for drug

administration. This preference is related with its easy access and non-invasive nature, which

improves patient compliance and, therefore, facilitates treatments. However, the poor water

solubility of several drug molecules and/or the risk of degradation throughout the

gastrointestinal tract, turn into impossible their oral administration. In this perspective, efforts

have been done in order to improve the oral bioavailability of poorly water-soluble drugs, by

means of developing new colloidal delivery carriers. Among these systems are lipid

nanoparticles, which have been showing promising results. The main reason for this is related

with the well-known conception that lipids promote oral drug absorption, because they undergo

the same physiological mechanisms of food lipid digestion. Lipid nanoparticles also offer unique

properties such as small size, large surface area, increased drug loading and stability

(especially for lipophilic drugs), possibility of controlled drug release, no toxicity and high

bioavailability.

Therefore, the first objective of the present work was to perform the production,

characterization and study of a medium chain fatty acid (MCFA) SLNs and MCFA/natural oil

NCLs prepared by sonication and employing a nonionic surfactant.

The second objective of the project was to study the obtained SLNs and NCLs as an

alternative system to improve the oral delivery of two anti-TB drugs, rifampicin and

pyrazinamide. Furthermore, β-carotene was also used as lipophilic model. Its delivery has a

great potential for food and cosmetic industry.

1

1. Introduction

Nanotechnology continues to play a growing and tremendous interest, both on academic

and industrial aspects. Such interest relies on the fact that it is now possible to manipulate

nanometer-length atoms and molecules in order to create, according to a bottom-up technology,

larger structures with outstanding properties. The conceptual underpinnings of

nanotechnologies were first laid out in 1959 by the physicist Richard Feynman in his lecture,

“There's plenty of room at the bottom” [1]. Feynman explored the possibility of manipulating

material at the scale of individual atoms and molecules, imagining the whole of the

Encyclopedia Britannica written on the head of a pin and foreseeing the increasing ability to

examine and control matter at the nanoscale. However, the term 'nanotechnology' was not

created until 1974, when Professor Norio Taniguchi of Tokyo Science University used it to refer

to the ability to engineer materials precisely at the nanometer level [2].

In the last decade, drug delivery research is clearly moving from the micro to the

nanometer scale. In simple terms, a drug delivery system is defined as a formulation or a device

that enables the introduction of a therapeutic substance in the body and improves its efficacy

and safety by controlling the rate, time, and place of release of this substance in the body. The

process of drug delivery includes the administration of the therapeutic product, the release of

the active ingredients by the product, and the subsequent transport of the active ingredients

across the biological membranes to the site of action [3].

The new technologies employed in drug discovery lead to find many new powerful

substances. However, the development of new drugs alone is not sufficient to ensure progress

in drug therapy. Poor water solubility and insufficient bioavailability of the new drug molecules

are main and common problems [4]. Therefore, there is an increasing need to develop a drug

carrier system that overcomes these drawbacks. This carrier system should have no toxicity

(acute and chronic), have a sufficient drug loading capacity and the possibility of drug targeting

and controlled release characteristics. It should also provide chemical and physical stability for

the incorporated drug. The feasibility of production scaling up with reasonable overall costs

should also be required [5,6].

Colloidal drug delivery systems, particularly those in the nanosize range, have been

increasingly investigated in the last years because they can fulfil the requirements mentioned

above[7–9]. Size reduction is one of the methods to increase the solubility and hence the

bioavailability of poorly-water soluble actives. Examples of such colloidal carriers are liposomes

[10], nanoemulsions [11], micelles [12–15], polymeric nanoparticles[16–18], lipid nanoparticles

[19,20], dendrimers [21–24] and drug nanocrystals [25–27]. Corresponding to the broad

diversity of colloidal carriers, there are many possible administration routes: dermal [28–31], oral

[32–36], parenteral [37,38], ocular [39], pulmonary [40–43] and intravenous [34,44]. According

to the aim of this thesis, the work will only focus along of the text on lipid nanoparticles. The

interested reader can find in the previous references high-quality reviews regarding the other

carriers.

2

1.1. Lipid Nanoparticles

Lipid nanoparticles were invented in the beginning of the nineties by two different

researchers and co-workers, regarding their different production methods: the R.H. Müller and

J.S. Lucks from Germany [45], and M.R. Gasco from Italy [46]. Nowadays, despite the number

of academic groups interested in the study of these systems have been increased, R.H. Müller

and co-workers are still the leaders of this research area [20].

In general, there are two types of lipid nanoparticles with a solid matrix, the solid lipid

nanoparticles (SLNs) and the nanostructured lipid carriers (NLCs), which differ in their inner lipid

structure (Figure 1).

Figure 1 – Schematic structure of solid lipid nanoparticle (a) and nanostructured lipid carrier (b).

SLNs consist of a solid lipid matrix that is solid at both room and body temperatures and

that are prepared in a similar manner to an oil-in-water (o/w) emulsion, except that the oil phase

of the emulsion is replaced by a solid lipid or a blend of solid lipids at room temperature [10].

Consequently, SLNs may be composed of a lipid or a mixture of lipids dispersed in an aqueous

phase at high temperature and if necessary stabilized with a surfactant or combination of

surfactants and then solidify at room temperature [10]. Particle size of SLNs range between 50-

1000 nm [10].

The incorporation of drugs in SLNs can be described by three different models: the

homogenous matrix model (in which drug is either molecularly dispersed or present as

amorphous clusters in the lipid matrix); the drug-enriched shell model (outer lipid shell

containing drug with lipid core) and the drug-enriched core model (drug core surrounded by lipid

layer or reservoir type system) [10,32]. The three different models are depicted in Figure 2.

3

Figure 2 - Models of drug incorporation in Solid Lipid Nanoparticles. From [32].

The morphological differences between those models depend on the properties of the

drug, on the matrix composition (in terms of lipids and surfactants), as well as on the method

selected for their production. In many cases, a mixture of the three types of systems is obtained

and, depending on the partition coefficient of the drug between the lipid and the aqueous

phases, the drug molecules can also be adsorbed onto the surface of the systems being

therefore not physicochemically protected by the matrix. The homogenous matrix model is

assumed for entrapped drugs that can show prolonged release from SLNs. The drug-enriched

shell model is obtained when phase separation occurs during the cooling process from the

liquid oil droplet to the formation of SLNs. The lipid precipitates first, forming an almost drug-free

lipid core. At the same time, the concentration of drug in the remaining liquid lipid increases

continuously. Finally, the compound-enriched shell crystallizes. The drug-enriched core model

is formed when the opposite mechanism as described for the former model occurs. In this case,

the drug precipitates first and the lipid shell formed around this core will have distinctly less

drug. This leads to a membrane-controlled release governed by Fick’s law of diffusion. This

model is formed when the drug concentration is close to its saturation solubility in the melted

lipid [10,32].

SLNs combine the advantages of early controlled drug delivery systems while minimise

their shortcomings. In general, a solid core offers many advantages in comparison to a liquid

core. Emulsions and liposomes usually show lack of protection of encapsulated drugs and drug

release as a burst (emulsions) or noncontrolled (from liposomes). SLNs allow for a controlled

release effect, whilst protect the drug against degradations, have good long term stability and

higher drug loading capacity. Polymeric nanoparticles also possess a solid matrix identical to

SLNs. However, SLNs can be manufactured using physiologically acceptable and

biodegradable lipid materials that have a GRAS (Generally Recognized as Safe) status and

require the use of lower amounts of organic solvents during production, which decreases the

toxicity [28]. Finally, and by contrast with liposomes and polymeric nanoparticles, SLNs have

low cost excipients and production techniques and are easily transferred to large scale.

Nonetheless, certain drawbacks have been associated with the use of SLNs such as:

SLNs dispersions contain high amount of water, drug loading capacity of SLNs are limited due

4

to crystalline structure of solid lipid, expulsion of encapsulated drug may take place during

storage due to formation of a perfect crystalline lattice especially when SLNs are prepared from

high purified lipid, drug release profile may change with storage time, polymorphic transitions

are possible, particle growth is also possible during the storage, and gelation of the dispersion

may take place during storage [47].

SLNs prepared from one highly purified lipid can crystallize in a perfect crystalline lattice

that allows very small space for the incorporation of drugs (Figure 1a). Lipids crystallize in high

energetic lipid modifications, α and β′, immediately after preparation of SLN. However, the lipid

molecules undergo a time-dependent restructuring process leading to formation of the low-

energetic modifications, βi and β, during storage. Formation of this perfect lipid crystalline

structure leads to expulsion of drug. Therefore, despite SLNs being interesting delivery

systems, relatively low drug loading capacity and potential expulsion of the drug during storage

led scientists to think about new strategies.

Consequently, a second generation of lipid nanocarriers, referred to as nanostructured

lipid carriers (NLCs) were developed at the turn of the 21st century [48]. NLCs matrices consist

of a less ordered lipid matrix with imperfections due to the mixtures of solid and liquid lipids

(Figure 1b) [38].

There are three types of NLC: the imperfect type, the multiple type, and the amorphous

type (Figure 3). The imperfect type is achieved by mixing solid lipids with small amounts of

liquid lipids. If higher amounts of oil are mixed with the solid lipid, a different type of

nanostructure is present. Here, the solubility of the oil molecules in the solid lipid is exceeded;

this leads to phase separation and the formation of oily nanocompartments within the solid lipid

matrix [48,49]. Many drugs show a higher solubility in oils than in solid lipids so that they can be

dissolved in the oil and still be protected from degradation by the surrounding solid lipids. This

type of NLC is called the multiple type, and can be regarded as an analogue to w/o/w emulsions

since it is an oil-in-solid lipid-in-water dispersion. Since drug expulsion is caused by continuing

crystallization or transformation of the solid lipid, this can be minimized by the formation of a

third type, the amorphous type. Here, the particles are solid but crystallization upon cooling is

avoided by mixing special lipids (e.g., hydroxyoctacosanylhydroxy-stearate and

isopropylmyristate) [48,49].

Figure 3 - Models of drug incorporation in Nanostructured Lipid Carriers. From [50].

5

In fact, the different NLC lead to the possibility of incorporating a high amount of drug

inside these systems, and moreover, control of drug release can still be observed because NLC

matrix is still solid. As a result, NLCs have several advantages such as: NLCs dispersions with

higher solid content can be produced, drug loading capacity is better than SLNs, drug release

profile can be easily modulated, drug leakage during storage is lower than SLNs, and

production of final dosage forms (e.g., tablets, capsules) is possible.

1.1.1. Lipid Nanoparticles as Oral Drug Delivery System

Oral route is the most preferred route for drug administration due to greater convenience,

less pain, high patient compliance, reduced risk of cross-infection, and needle stick injuries.

Major portion of the drug delivery market is occupied by oral drug delivery systems. However,

oral drug delivery is continuously looking for new strategies due to factors such as low drug

solubility, poor gastrointestinal (GI) absorption, rapid metabolism, high fluctuation in the drug

plasma level, and variability due to food effects. In addition, the acidic environment and the

presence of several enzymes in distinct parts of the GI tract increase the risk of occurrence drug

degradation [51]. These factors may cause disappointing in vivo results leading to failure of the

conventional delivery systems [52].

Intake of a high-fat meal leads to prolongation of GI tract residence time, stimulation of

biliary and pancreatic secretions, stimulation of lymphatic transport, enhancement of intestinal

wall permeability, reduction of metabolism and efflux activity, and alteration in mesenteric and

liver blood flow, which significantly contribute to improve oral bioavailability of drugs [53,54].

Therefore, lipid-based delivery systems may reduce the inherent limitations of slow and

incomplete dissolution of poorly soluble drugs and facilitate the formation of solubilised phase

from which absorption may occur [55,56].

A normal healthy adult GI tract is able to daily hydrolyse about 100-140 g of dietary lipids

(mainly in the form of triglycerides). Despite the exact body mechanisms for lipid processing

remains unclear, the procedure can be divided in digestive, absorption and systemic blood

uptake phases. Lipids digestion generally begins in the stomach where triglycerides are

hydrolysed to diglycerides and fatty acids by the acid-stable lipases such as lingual lipase and

gastric lipase. Acid lipases have a greater affinity for medium chain triglycerides when

compared with long chain triglycerides and do not hydrolyse phospholipids or cholesterol esters.

Acid lipases are also inhibited by long chain fatty acid digestion products, (which are mostly

protonated at gastric pH), and therefore digestion via acid lipases accounts for only

approximately 10 to 30% of the overall hydrolysis of ingested triglycerides in food. Afterwards,

gastric contents reach the duodenum, the first section of small intestine, where the presence of

lipids stimulates both productions of lipase/co-lipase enzymes by pancreas and bile salts

(phospholipids and cholesterol) by the gall bladder. Bile salts adhere to the surface of emulsion

droplets promoting the lipase/co-lipase action and originating free fatty acids and colloidal

6

species like micelles, mixed micelles, vesicles. Micelles are composed by surfactant molecules

which self-assemble in aqueous solution above a determinate concentration, the so-called

critical micellar concentration (CMC). Mixed micelles are similar to micelles but are composed of

various surfactants molecules. Vesicles are formed by the self-assembling of insoluble

phospholipids. Absorption occurs mostly in the small intestine where occurs the passage of

substances directly to systemic circulation, or firstly to lymphatic circulation and subsequently to

blood [57,58].

Concerning the typical lipid nanoparticles triglyceride-based composition, is expected that

after oral administration, they undergo similar mechanisms of food-ingested lipids. The size of

nanoparticle is an important factor for uptake into the epithelial of GI. Intestinal cells cannot

absorb nanoparticles larger than 400 nm [59]. Furthermore, lipid nanoparticles have adhesive

properties, which permit their adherence to the enterocytes (intestinal epithelial cells) surface.

Therefore, the drug release from the nanoparticles is immediately followed by direct absorption

within the enterocytes. In parallel, the presence of lipid nanoparticles in the duodenum promotes

both lipase/co- lipase activities and bile salts secretion. The former hydrolyse the triglycerides in

monoglycerides and fatty acids forming micelles, which undertake (i.e. re-solubilise) the drug

meanwhile it is released during the degradation of the nanoparticles. Additionally, the bile salts

interact with micelles and form mixed micelles. Subsequently, drug is absorbed together with

these colloidal species, by one or more of the following transport mechanisms (Figure 4):

transcellular, which is elected by small lipophilic molecules and involve the passive passage

throughout the intestinal epithelial cells; paracellular implies the passive passage through the

aqueous pores existing between enterocytes (tight junctions) and, therefore, is the selected

path by hydrophilic drug molecules; active carrier mediated require the association of drug

molecules with a specific transporter or carrier, which undertake the passage through

enterocytes; receptor mediated comprise cell internalization of drug molecules by means of

processes like endocytosis, phagocytosis and pinocytosis.

After crossing intestinal epithelia, the most part of drug molecules pass directly to the

hepatic portal vein, which carries them to the liver and afterwards to the systemic circulation.

This process constitutes a problem when drugs undertake first-pass metabolism, since they can

be inactivated before reach their local of action. Nonetheless, highly lipophilic drugs (log P > 5)

tend to entry mostly by lymphatic circulation, when comparing to portal circulation, which avoids

the risk of occurring liver first-pass metabolism. Hence, lipid can augment lymphatic uptake of

several drugs, especially lipophilic drugs or large molecular weight macromolecules.

Furthermore, lymphatic capillaries are significantly more permeable to nanoparticles than the

blood capillaries.

However, lymphatic absorption depends on the length of the fatty acid chains. Small- and

medium-chain lipids across the enterocytes by diffusion and enter directly to systemic

circulation, while long-chain lipids pass first to endoplasmic reticulum where they are re-

esterified into triglycerides and associated with lipoproteins. Following they pass to lymphatic

circulation and finally enter into systemic circulation.

7

Figure 4 - Various mechanisms of enhancement of drug bioavailability in the presence of lipids.

Oral administration of SLNs is possible as aqueous dispersion [60] or alternatively

transformed into a traditional dosage forms such as tablets, pellets, capsules, or powders in

sachets [58]. Since the stomach acidic environment and high ionic strength favour the particle

aggregation, aqueous dispersions of lipid nanoparticles might not be suitable to be administered

as dosage form. In addition, the presence of food will also have a high impact on their

performance [5]. The packing of SLNs in a sachet for redispersion in water or juice prior to

administration will allow an individual dosing by volume of the reconstituted SLNs. For the

production of tablets, the aqueous SLNs dispersions can be used instead of a granulation fluid

in the granulation process. Alternatively, SLNs can be transferred to a powder (by spray-drying

or lyophilisation) and added to the tableting powder mixture. In both cases, it is beneficial to

have a higher solid content to avoid the need of having to remove too much water. For cost

reasons, spray drying might be the preferred method for transforming SLNs dispersions into

powders, with the previous addition of a protectant [48]. For the production of pellets, the SLNs

dispersion can be used as a wetting agent in the extrusion process. SLNs powders can also be

used for the filling of hard gelatine capsules.

Several drugs (hydrophobic and hydrophilic) have been incorporated in lipid

nanoparticles for oral administration. Table 1 shows some of these drugs encapsulated in SLNs

and NLCs examples and their potential benefits for application in oral delivery.

8

Table 1 – Examples of different Drug-Loaded Lipid Nanoparticles and their potential benefits for

application in oral administration route.

Drug Lipid

Nanoparticle Relevant effects Reference

Amphotericin B SLN

Improvement of oral bioavailability;

Drug protection;

In vivo drug controlled release;

[61]

Apomorphine SLN


Successful targeted drug to the local of

therapeutic action (brain striatum);

[62]

Camptothecin SLN Body distribution;

Sustained release and tissue targeting; [63]

Candesartan

cilexetil SLN Improvement of oral bioavailability; [64]

Carvedilol SLN

Enhancement of oral bioavailability;

Lymphatic uptake;

Bypass hepatic first-pass metabolism;

[65]

Clozapine SLN Improvement of oral bioavailability;

Tissue distribution; [34]

Cryptotanshinone SLN

Enhancement of GI absorption;


Change in metabolism;

[66]

Curcumin SLN Improvement of oral bioavailability

Prolonged release; [67]

Cyclosporine A SLN Low variation in bioavailability; [68]

Digoxin SLN Enhancement of GI absorption

Improvement of oral bioavailability; [69]

9

Edelfosine SLN

High accumulation of drug in the local of

therapeutic action (brain);

In vitro antiproliferative effects on glioma cells;

In vivo reduction of tumor growth;

[70]

Etoposide NLC


In vitro antiproliferative effects on lung

carcinoma cells;

[71]

Fenofibrate SLN Improvement of oral bioavailability [72]

Insulin Lectin-

modified SLN

Protection of insulin degradation by enzyme;



[33]

Insulin SLN Enhancement of GI absorption;

Importance of delivery site; [73]

Lopinavir SLN


Effective target of the drug to the local of

therapeutic action (CNS);

Prolong the blood circulation time of the drug;

[74]

Lovastatin NLC

Enhancement of encapsulation efficiency;


Improvement of stability in GI environment;

[75]

Melatonin SLN Sustained delivery [76]

Methotrexate SLN


Lymphatic transport;


[23]

N3-O-toluyl-

fluorouracil Cationic SLN



N3-O-toluyl-

fluorouracil Anionic SLN Improvement of intestinal transport; [60]

Nitrendipine SLN Improvement of oral bioavailability;

Reduction of first-pass metabolism; [78]

10

Norfloxacin SLN Improvement of oral bioavailability;

Prolong the plasma drug level; [79]

Ofloxacin SLN


In vitro drug controlled release;

In vitro enhanced antibacterial activity;

[80]

Otcadecylamine SLN


Prolonged release;

Lymphatic transport;

[81]

Paclitaxel SLN

.Improvement of oral bioavailability;

In vitro drug controlled release;

In vivo safety properties;

[82]

Pentoxifylline SLN Improvement of oral bioavailability;

Reduction of first-pass metabolism; [83]

Praziquantel SLN


Improvement of bioavailability;

Prolonged systemic circulation;

[84]

Puerarin SLN


Increase of drug concentrations in tissues,

especially for the target organs (heart and brain);

[85]

Quercetin SLN Enhancement of GI absorption;


Repaglidine SLN Improvement of oral bioavailability;

In vivo safety properties; [87,88]

Rifampicin,

Isoniazid, and

Pyrazinamide

SLN Prolonged release; [89]

Risperidone SLN In vitro safety properties;

In vitro drug controlled release; [36]

Simvastatin SLN Improvement of oral bioavailability; [90]

11

Spironolactone SLN Improvement of oral bioavailability; [91]

Tretinoin SLN In vitro drug controlled release; [92]

Vinpocetine NLC Improvement of oral bioavailability; [93]

Vinpocetine SLN Enhancement of GI absorption;


α-Asarone SLN



Improvement of tissue uptake and distribution;

[95]

γ -Tocotrienol SLN Improvement of oral bioavailability. [96]

1.1.1.1. Toxicological Concerns

Lipid nanoparticles are generally made of physiological and GRAS excipients and

therefore, metabolic pathways exist decreasing the risk of acute and chronic toxicity. Their

degradation is relatively fast in non-toxic compounds that are easily eliminated through

physiologic metabolic pathways. The lipid matrix degradation occurs mostly by lipases whereas

only a minor part is degraded by non-enzymatic hydrolytic processes [97]. Nonetheless, even

for biodegradable nanoparticles, the use of high concentrations of the carriers can lead to

toxicological concerns. Therefore, the fate of the carriers in the body should be clarified.

The human immune system, by means of macrophages, recognize all external

nanoparticles as hostile matter and quickly phagocyte and clearance them from the body.

Nonetheless, these immunological specialized cells are present in limited areas of the body

(e.g. lungs), which decrease the risks of interference with oral administered systems.

Furthermore, the nanoparticles with sizes below to 100 nm can be internalized by all body cells

[20,98]. Regarding the typical lipid nanoparticles sizes of 100-300 nm (size above 100 nm),

which means that their cellular uptake is not expected and, therefore, no significant toxicological

concerns exist [25].

12

Toxicological studies should be first performed in vitro, using cell models that mimic the

body conditions, in order to minimize the number of animal studies and to have an idea of the

cytotoxicity of the systems in an early stage. Nonetheless, in vivo studies must always be

performed before pass to human clinical trials, since in vitro studies sometimes use short time

periods and small concentration ranges, which not allow for realistic conclusions about the

cytotoxicity of the nanocarrier systems [99].

Silva et al. [36] studied the toxicity of SLN and risperidone loaded SLNs with Caco-2 cells

by (4,5-dimthylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. The Caco-2 cell line

is a human colon epithelial cancer cell line often used as a model to mimic the GI tract

conditions for toxicological and pharmacological studies [100]. MTT assay evaluates the

mitochondrial function as a measurement of cell viability. Therefore, only live cells could react

with the MTT reagent and the cell viability is detected in a very early stage. The results suggest

that all formulations evaluated are biocompatible with Caco-2 cells and well tolerated by the GI

tract. Similar results have been reported elsewhere [101,102]. The amount of viable cells after

SLNs exposure was performed by the MTT assay with Caco-2 cell models. Other authors have

also reported that SLN show biocompatibility, which increase their attractiveness for drug-

delivery applications [101].

Several studies have reported that lipid nanoparticles systems are well tolerated and

demonstrate low cytotoxicity, compared with other conventional nanocarriers [101,103]. Lipid

carriers prepared with several lipids and emulsifying agents did not exhibit substantial cytotoxic

effects in vitro. SLN prepared up to concentrations of 2.5% lipid do not exhibit any cytotoxic

effects in vitro [104]. In fact, it has been shown that even concentrations higher than 10% of lipid

led to a viability of 80% in cultures of human granulocytes [105]. In contrast, some polymeric

nanoparticles showed complete cell death at concentrations of 0.5%. It can be assumed that the

cytotoxicity of the SLNs can be mainly attributed to emulsifiers and preservative compounds

which are used in the production of these systems. Additionally, depending on the route of

administration, the toxicity requirements of the nanoparticles systems can change, being less

for dermal, moderate for oral and limited for intravenous administration [20].

In conclusion, from the data obtained until now, the lipid nanoparticles formulations

appear to fulfil the essential prerequisite to the clinical use of an oral colloidal carrier that means

low cytotoxicity.

13

1.1.2. Preparation Techniques for Lipid Nanoparticles

Since lipid nanoparticles dispersions at high temperatures are miniemulsions,

miniemulsions preparation techniques can be applied or modified for lipid nanoparticles

production. A miniemulsion, also referred as nanoemulsion or ultrafine emulsions, compose a

particular class of emulsions consisting of colloidal dispersions with a droplet size between 20-

500 nm. The physical appearance of nanoemulsion is transparent or bluish for the smallest

droplet sizes between 20–100 nm, or milky for sizes up to 500 nm [106]. Nanoemulsions are

non-equilibrium systems and cannot be formed spontaneously. Nanoemulsion can be produced

through two ways. There is either high-energy emulsification method or low-energy

emulsification method. High-energy emulsification method includes high-pressure

homogenization, sonication or high-shear mixing, whereas condensation method such as phase

inversion temperature (PIT) is an example of low-energy emulsification method [107].

As several techniques have been developed for the production of lipid nanoparticles,

resulting in different particles sizes and shapes, some parameter must be considered in order to

choose the suitable preparation method. The size can influence the pharmacological properties

of the particles, but it is not the unique parameter considered to compare the various

techniques. Toxicological issues are also very important. The materials used must be

biocompatible and biodegradable, while the use of solvents can be a relevant drawback, since

they can remain in traces in the final product. From a technological point of view, the possibility

to scale up the process is very important, but also the feasibility of the method is relevant. In

fact, the use of expensive and complex machine can hamper the production on lab scale.

Finally, the drug entrapment is very important. Nowadays more and more complex molecules

are entrapped within lipid nanoparticles. These molecules have different physical and chemical

properties (solubility, hydrophobicity, etc.) and stability issues (temperature, pH, etc.). The

chosen preparation technique should be the most appropriate to enhance drug loading and

encapsulation efficiency within the nanoparticles, without hampering the chemical stability of the

molecule itself.

The following sections describe different existing approaches for SLNs and NLCs

formulations. However, in some instances combination of different methods has been utilized to

prepare the lipid nanoparticles.

1.1.2.1. High Pressure Homogenization Technique

Müller and Lucks were the first to prepare SLNs by applying high pressure

homogenization (HPH) technique [108]. It makes use of high pressure homogenizer which is

accessible from several manufacturers. In this technique, melted lipid solution is forced at a high

pressure (100-2000 bar) through a narrow gap of few microns. The resulting high shear stress

and cavitation forces decrease the particle size. A homogeneous dispersion with narrow size

14

distribution is desirable to increase the physical stability of the aqueous dispersion. If the

particles localized at different positions in the dispersion volume, experience different forces

then the degree of particle disruption will vary. There are two basic production methods to HPH:

the hot and the cold homogenization techniques. In both the techniques, the lipid contents are in

the range of 5-10%, but even higher concentration of lipid (40%) can be homogenized to

nanodispersion [109].

1.1.2.1.1. Hot Homogenization Technique

In this method, the drug is first dissolved or solubilized in the lipid being melted at 5-10ºC

above its melting point. The drug-containing melt is dispersed under stirring in a hot aqueous

surfactant solution of identical temperature [110]. The pre-emulsion is then passed through a

high pressure homogenizer (e.g. Micron LAB40) for 3 to 5 cycles and applying a pressure of

about 500-1500 bar [111]. The obtained nanoemulsion is cooled down to room temperature or

lower. The lipid recrystallizes and leads to SLNs. Homogenization pressure and the number of

cycles should not be higher than that required to achieve the desired effects because this

increases the cost of production and the chances of metal contamination as well as in some

cases it would result in increase in particle size due to aggregation as a result of the high

surface free energy of the particles [112]. This technique is performed at a high temperature

and thus cannot be used for temperature sensitive drugs. So, to avoid heat-accelerated drug

degradation, the length of time for drug exposure should be shortened [10]. This method is also

not suitable for hydrophilic drugs because they would partition between the melted lipid and

aqueous phase during hot homogenization [10]. Either medium scale or large scale production

is possible for SLNs by HHT and it is the most extensively technique for the preparation of

SLNs.

1.1.2.1.2. Cold Homogenization Technique

In this process, the drug is also dissolved or solubilized in lipid being melted at 5-10ºC

above its melting point. The mixture is rapidly cooled down with help of liquid nitrogen or dry ice.

Rapid cooling procedure helps in the homogeneous distribution of the active ingredient. This

solidified mixture is milled to about 50-100 μm particles using a ball or mortar mill and followed

by dispersion of the lipid microparticles in a cold surfactant aqueous solution to obtain a

suspension. This suspension is then passed through a high pressure homogenization at or

below room temperature. Here, the cavitation forces are strong enough to break the lipid

microparticles to SLNs. The cold homogenization technique reduces the chances of drug

degradation induced by temperature and thus thermo label drugs can be used. Since the solid

lipid is milled the complexity arising due to lipid modification can be avoided [112]. Chances of

15

drug distribution into the aqueous phase are limited and hence this method can be used for

hydrophilic drugs as well as lipophilic drugs. Lipid nanoparticles prepared via this technique

possess a slightly larger particle size and polydispersity when compared to the ones obtained

by hot homogenization technique, using the same lipid at similar homogenization parameters

(pressure, temperature and the number of cycles) but a higher number of homogenization

cycles can be applied to reduce the particle size [10].

1.1.2.2. Microemulsion Technique

Gasco and co-workers were the first to develop SLNs based on the dilution of

microemulsions [46]. Microemulsions are thermodynamically stable, clear and isotropic mixtures

usually composed of an oil or lipid, emulsifier and/or co-emulsifier and water. In this process,

the solid lipids are melted above their melting point. Separately, a mixture of emulsifier, co-

emulsifier and water is heated at same temperature. Both the lipid and the aqueous phase

containing the emulsifier are mixed in appropriate ratios and stirred in order to produce a

microemulsion. The hot microemulsion is then diluted with cold water (2-8°C) while stirring. The

ratio of the hot microemulsion to cold water is usually in the range of 1:10 to 1:50 [113]. It has

been noted that a droplet structure is already present in the microemulsion and therefore, no

external energy is required to achieve the small particle size. When the microemulsion is diluted

by cold water, the lipid droplets solidify as the temperature decreases. The temperature gradient

and pH value determine the quality of the particles in addition to the composition of the

microemulsion. Subsequently, the produced SLNs are washed three times with distilled water

and followed by membrane filtration in order to remove any unwanted bigger lipid particle. Due

to the dilution of microemulsion the concentrations of particle content are below 1% and,

therefore, large amount of water has to be removed either by ultrafiltration or by lyophilisation in

order to increase the particle concentration. The major limitation of this technique is its

sensitivity to minor changes in composition or thermodynamic variables, which can lead to

phase transitions. Lack of robustness of the microemulsion technique can lead to high

production costs. Moreover, lipids solidification shifts the system to a thermodynamically

unstable state. The high concentration of surfactants used may produce toxicity.

Ultracentrifugation, ultrafiltration or dialysis can be applied to remove excess surfactants [113].

1.1.2.3. Ultrasonication Technique

In this technique, solid lipids are melted at 5–10°C above their melting points and drug is

dissolved or dispersed in melted lipids. Then a hot aqueous surfactant solution (preheated at

the same temperature) is added to the drug-lipid melt and homogeneously dispersed by a high

shear mixing device. The coarse hot o/w emulsion obtained is ultrasonicated using a probe

16

sonicator till the desired sized nanoemulsion is formed. Finally, lipid nanoparticles are obtained

by allowing hot nanoemulsion to cool down to room temperature.

Two mechanisms are proposed for ultrasonic emulsification. First, the application of an

acoustic field produces interfacial waves resulting in the dispersion of the oil phase in the

continuous phase in the form of droplets [114]. Secondly, the application of ultrasound induces

acoustic cavitation causing the formation and subsequent collapse of microbubbles by the

pressure fluctuations of a simple sound wave, which creates extreme levels of highly localized

turbulence. Therefore, the turbulent micro-implosions break up primary droplets into sub-micron

size [115].

This technique is a simple and reproducible way to prepared nanoparticles without the

need of organic solvents or any sophisticated instruments and has the potential to easily scale

up for large scale production. However, metallic contamination of the product may occur during

sonication by probe sonicator [116].

1.1.2.4. Solvent Emulsification Evaporation Technique

Sjöström and Bergenståhl were the first to describe the production of SLNs by solvent

emulsification-evaporation technique [117]. The solid lipid is dissolved in a water immiscible

organic solvent (e.g. cyclohexane, dichloromethane, toluene, chloroform, etc.) and the drug is

dissolved or dispersed in the solution [118]. This organic phase containing the drug is emulsified

in an aqueous surfactant solution by mechanical stirring. The organic solvent is then removed

from the emulsion under mechanical stirring or reduced pressure (40-60 mbar) (e.g. rotary

evaporator) [118]. Lipid nanoparticle dispersion is formed by the precipitation of the lipid phase

in the aqueous surfactant medium. The mean particle size depends on the concentration of lipid

in organic phase. Very small particle size can be obtained with low lipid load (5%) related to

organic solvent. Aggregation of the particles can be avoided by removing the solvent at a faster

rate [10]. Thermo labile drugs can be incorporated via this technique as it avoids thermal stress.

Trace amounts of organic solvent remaining in the final product can potentially create toxicity

problems. A large quantity of water has also to be removed during the final step of the

formulation by means of ultrafiltration or evaporation [119,120].

1.1.2.5. Double Emulsion Technique

The double emulsion (w/o/w) method is based on solvent emulsification–evaporation

method [113]. This method is mainly for the production of lipid nanoparticles loaded with

hydrophilic drugs. In this case, drug is firstly dissolved in aqueous solvent (inner aqueous

phase) and then is dispersed in lipid containing emulsifier/stabilizer to produce primary emulsion

(w/o). A double emulsion (w/o/w) is formed after addition of an aqueous solution with a

17

hydrophilic emulsifier followed by stirring. Each emulsification step results in a highly

polydisperse droplet distribution, exacerbating the polydispersity of the final double emulsion.

Thus, lipid nanoparticles formed from double emulsions technique are, by nature, poorly

controlled in both size and structure.

1.1.2.6. Solvent Emulsification Diffusion Technique

Quintanar-Guerrero et al. were the first to describe this technique for the preparation of

polymeric nanoparticles [121]. Recently this technique has been modified by various research

groups for the preparation of lipid nanoparticles [122,123]. In solvent emulsification diffusion

technique, the lipid is dissolved in a partially water miscible solvent (e.g. benzyl alcohol,

isobutyric acid, or tetrahydrofuran) which is previously saturated with water at room temperature

or at a controlled temperature in order to ensure the initial thermodynamic equilibrium [113]. The

mixture is then emulsified in an aqueous solution of a surfactant by mechanical stirring at the

temperature used to dissolve the lipid producing an o/w emulsion. This o/w emulsion is then

diluted with excess water, in typical ratio ranges from 1:5 to 1:10 [113], at a controlled

temperature which causes the diffusion of the solvent into the external phase and subsequent

precipitation of the lipid nanoparticles. The diffused solvent can be removed either by vacuum

distillation or ultrafiltration. The concentration and the nature of the lipid and surfactant, stirring

rate and the processing temperature are critical variables in this technique. This technique

avoids the necessity to melt the lipid which is very useful for preparation of protein and peptide-

loaded lipid nanoparticles.

1.1.2.7. Solvent Injection Technique

The basic principle of the solvent injection method is similar to the solvent emulsification

diffusion technique. In case of solvent injection method, lipids are dissolved in a water-miscible

solvent (e.g., acetone, isopropanol, and methanol) or water-miscible solvent mixture and quickly

injected into an aqueous solution of surfactants through an injection needle [113]. The

advantages of this method are the easy handling and fast production process without

technically sophisticated equipment (e.g., high-pressure homogenizer). However, the main

disadvantage is the use of organic solvents. The resulted dispersion is filtered through a filter

paper in order to remove any excess lipid [113]. The presence of emulsifier within the aqueous

phase helps to produce lipid droplets at the site of injection and stabilize lipid nanoparticles until

solvent diffusion was complete by reducing the surface tension between water and solvent.

18

1.1.2.8. Phase Inversion Temperature Technique

The phase inversion temperature (PIT) method is commonly used for the preparation of

nanoemulsions. The PIT concept uses the specific ability of some polyethoxylated surfactants to

modify their affinities for water and oil as a function of the temperature. In the PIT nanoemulsion

preparation method, the use of such surfactant type leads to an emulsion inversion from o/w

macroemulsion to a w/o emulsion when temperature is increased above the PIT, and to the

formation of an o/w nanoemulsion when the temperature is next lowered below the PIT [124].

Recently it has been adapted for the preparation of SLN. In this case two main components are

used: an oil phase, constituted by solid lipids and non-ionic surfactants and an aqueous phase

containing NaCl. The aqueous phase and the oil phase are separately heated above the PIT;

then the aqueous phase is added dropwise, at constant temperature and under agitation, to the

oil phase, in order to obtain a w/o emulsion. The mixture is then cooled to room temperature

under slow and continuous stirring. At the PIT, the turbid mixture becomes clear, then below the

PIT an o/w nanoemulsion is formed, which turns in lipid nanoparticles below the lipid melting

point [125].

1.1.2.9. Microchannel/Microfluidic Technique

A novel microchannel system with a cross-shaped junction was developed by a main

microchannel and two branches as shown in the Figure 5. The lipid dissolved in a water-

miscible organic solvent is passed through the main channel, while simultaneously an aqueous

surfactant solution is introduced into the branches. These two liquids met together at the cross-

shaped junction and passed along the main channel. The solvent diffused from the lipid solution

stream into the aqueous phase, which resulted in the local supersaturation of lipid and thus led

to the formation of lipid nanoparticles. The size of lipid nanoparticles prepared in the

microchannel system was influenced either by velocities of the lipid and aqueous phases and by

lipid and surfactant concentrations [113]. This technique allows precise control of the outer and

inner drop sizes as well as the number of droplets encapsulated in each larger drop [113].

However, in a continuous production process, lipid nanoparticles contained in the liquid may

block or deposit inside the microchannel, which could result in the break down and failure of

particle preparation [113].

Figure 5 - Schematic diagram of lipid nanoparticles formation in the microchannel system. From [113]

19

1.1.3. Characterization of Lipid Nanoparticles

Characterization of lipid nanoparticles is a challenge due to the small size of these

colloidal carriers and complexity of the system. Numerous parameters need to be considered,

such as mean particle size, zeta potential, drug association efficiency, degree of lipid

crystallinity and lipid modification. For each parameter can be applied several techniques.

However, only methodologies selected in this work will be further described.

1.1.3.1. Measurement of Particle Size

Particle size might be determined by dynamic light scattering (DLS), optical single particle

sizing (OSPS), laser diffraction (LD), transmission electron microscopy (TEM), scanning

electron microscopy (SEM), atomic force microscopy (AFM), scanning tunnelling microscopy

(STM) and freeze fracture electron microscopy (FFEM) [112,113].

1.1.3.1.1. Dynamic Light Scattering

Dynamic Light Scattering (DLS) also referred as Photo Correlation Spectroscopy (PCS) is

a technique used to determine the mean particle size and the width of the particle size

distribution expressed as polydispersity index (PDI). The particle size is the diameter of the

sphere that diffuses at the same speed as the particle being measured.

The measurement using DLS is based on the light scattering phenomena in which the

statistical intensity fluctuations of the scattered light from the particles in the measuring cell are

measured. These fluctuations are due to the random movement of the particles in the

dispersion medium. This movement is called Brownian motion.

Usually a DLS device consists of a laser light which illuminates a small volume of the

sample composed by a dilute suspension of particles. The light scattered from these particles is

collected by a lens and its intensity is measured by a photomultiplier at a certain angle (90º or

173º). The diffusion rate of the particles depends on their size (at a known fluid viscosity and

temperature). Hence, the size of these particles can be calculated from the rate of fluctuation of

the scattered light intensity. When the suspended particles are small, they diffuse relatively fast,

and the fluctuations in the scattered light are rapid. On the other hand, if the particles are large,

they move slowly, and the fluctuations in the scattered light are slow. The detected intensity

signals are used by a correlator to calculate the auto-correlation function G(τ). A correlator is

basically a signal comparator designed to measure the degree of similarity between two signals,

or one signal with itself at varying time intervals. From the decay of correlation function, the

diffusion coefficient (D) of the particles is obtained. Once the diffusion coefficient is known, the

20

hydrodynamic diameter of a spherical particle can be calculated applying the Stokes-Einstein

equation (Eq.1),

d(H) = 𝐾𝑇

3πηD Eq.1

where, d(H) is the hydrodynamic diameter, D is translational diffusion coefficient which

measures the velocity of the Brownian motion, k is the Boltzmann’s constant, T is the absolute

temperature and η is the viscosity of the solution.

As it was previously mentioned, small particles diffuse faster than large ones, causing a

stronger fluctuation in the scattering signal and a more rapid decaying G(τ) (Figure 6). For a

monodisperse particle population, G(τ) is a single exponential, but if more than one size of

particles is present the function is polyexponential. Deviation from a single exponential is used

to calculate the PDI, which is a measure of the width of the size distribution. The PDI value is

zero when a monodisperse particles population is measured. PDI values of around 0.10-0.30

indicate a relatively narrow distribution, values of 0.5 and higher are obtained in case of very

broad distributions [126].

Figure 6 -Correlation differences between small and large particles. From [126].

1.1.3.2. Measurement of Zeta Potential

Almost all particles in contact with a liquid acquire an electric charge on their surface. The

development of a nett charge at the particle surface affects the distribution of ions in the

surrounding interfacial region, resulting in an increased concentration of counter ions close to

the surface. Thus an electrical double layer exists around each particle (Figure 7). The liquid

21

layer surrounding the particle has an inner region called the Stern layer, where the ions are

strongly bound and an outer and diffuse region, where the ions are less firmly attached. Within

the diffuse layer there is an imaginary boundary inside which the ions and particles form a

stable entity. When a particle moves, ions within the boundary move with it, but any ions beyond

the boundary do not move with the particle. This boundary is called the surface of hydrodynamic

shear or slipping plane. Zeta potential (ζ) is the potential that exists at this boundary and it is a

parameter which is very useful for the assessment of the physical stability of colloidal

dispersions [126].

Figure 7 - Zeta-potential of a nanoparticle in solution. From [126].

Charged particles suspended in the electrolyte are attracted towards the electrode of

opposite charge, when an electric field is applied. Viscous forces acting on the particles tend to

oppose this movement. When equilibrium between these two opposing forces is achieved, the

particles move with a constant velocity. This velocity of the particle per unit electric field strength

(E) is called the electrophoretic mobility (μ), which is expressed in micrometers per second per

volt per centimeter (μm/s)/(V/cm). Zeta Potential can be measured by electrophoretic mobility

through Henry’s equation (Eq. 2),

μ = 2εζf(ka)

3η Eq. 2

where η is the viscosity of the dispersion medium, ε the permittivity of the environment (the

dielectric constant) and f(ka) is the Henry’s function. Electrophoretic determinations of zeta

22

potential are most commonly made in aqueous media and moderate electrolyte concentration.

In this case, f(Ka) is 1.5 and is referred to as the Smoluchowski approximation. Non-aqueous

meassurements generally use the Huckel approximation and f(Ka) is 1 [126].

The storage stability of a colloid system can be predicted by the measurement of the zeta

potential. It is currently accepted that absolute zeta potentials values higher than 30 mV are

required for full electrostatic stabilization; potentials between 5 and 15 mV are in the region of

limited flocculation; and lower than 3 mV of maximum flocculation. As a result, particle

aggregation is less expected to occur for charged particles (high zeta potential) due to electric

repulsion [113].

Nanoparticles with a zeta potential between -10 and +10 mV are considered

approximately neutral, while nanoparticles with zeta potentials of greater than +30 mV or less

than -30 mV are considered strongly cationic and strongly anionic, respectively. Since most

cellular membranes are negatively charged, zeta potential can affect a nanoparticle's tendency

to permeate membranes, with cationic particles generally displaying more toxicity associated

with cell wall disruption. Positive charge possesses a membrane destabilizing and

concomitantly destructive effect resulting from an interaction of positive charge and negative

charge of membrane. Thus, positively charged lipids are not approved by Food and Drug

Administration (FDA) for clinical use [127].

1.1.3.3. Encapsulation Efficiency

According to literature, the association efficiency of a drug in SLNs is most frequently

assessed indirectly, meaning that the aqueous phase is separated from the lipid particles and

subsequently drug content of the aqueous phase is determined [113]. The separation of the

SLNs from the aqueous phase can be done by e.g. ultrafiltration, ultracentrifugation and gel

separation. The amount of drug measured in the aqueous phase is considered the amount of

drug not incorporated. The following equation (Eq. 3) is used to calculate the association

efficiency:

EE% = 𝑇𝐷−𝐹𝐷

TD × 100 Eq.3

where TD is total drug weighted at the beginning and FD is the free drug dissolved in aqueous

medium.

1.1.3.4. Morphology

Images of lipid nanoparticles could be achieved through several microscopic techniques,

such as the scanning electron microscopy (SEM), transmission electron microscopy (TEM),

23

atomic force microscopy (AFM), CryoSEM and CryoTEM [112,113]. These techniques are very

useful since allow the visualization and measurement of particles much smaller than 1 μm.

1.1.3.4.1. Transmission Electron Microscopy

TEM utilizes energetic electrons to provide morphologic, compositional and

crystallographic information of samples. At a maximum potential magnification of 1 nm, TEM is

the most powerful microscope, producing high-resolution and two-dimensional images, allowing

the investigation of micro- and nanostructures. Going through the several parts of the TEM

system, first an electron gun generates a high energy electron beam by accelerating electrons

emitted from a cathode. This acceleration voltage, usually greater than 100 kV, determines the

microscope’s resolution. After the accelerator, there are condenser lenses to control beam

diameter and convergence angles of the incident beam on a specimen. The TEM has three

lenses to ensure good magnification capability, and the intermediate lens is used to switch the

TEM between an image mode and a diffraction mode. TEM specimens must be thin foils (about

100 nm) because they should be able to transmit electrons, and this thin specimen is mounted

in a 3 mm diameter grid. The specimen preparation depends on the type of sample.

1.1.3.5. Lipid Crystallinity

Lipid crystallinity and polymorphic changes during storage are strongly related with drug

incorporation and release rates [112,113]. Due to the small size of the SLN and the presence of

surfactants, lipid crystallization and modification changes can be highly retarded. Consequently,

special attention should be paid on the characterisation of such parameters. Lipid crystallinity

and polymorphic forms are mainly investigated by differential scanning calorimetry (DSC) and

X-ray scattering techniques [112,113].

DSC is a thermal analytical technique commonly used to characterize the physical state,

the degree of crystallinity, polymorphism, crystal ordering, eutectic mixtures or glass transition

processes in SLNs dispersions [112,113]. Additionally, DSC experiments are useful to

understand drug and lipid interactions within SLNs.

24

1.2. Tuberculosis – Review

Tuberculosis (TB) is an infectious disease caused by bacilli belonging to the

Mycobacterium tuberculosis complex [128] and this complex consists of seven species

including Mycobacterium tuberculosis, Mycobacterium canetti, Mycobacterium africanum,

Mycobacterium pinnipedii, Mycobacterium microti, Mycobacterium caprae and Mycobacterium

bovis [129].

TB is predominantly a disease of the lung, with pulmonary TB accounting for about 70%

of the cases. Extra-pulmonary disease sites include lymph nodes, bone, and meninges [130] .

The bacilli remain viable in the air for a long time and, eventually, they are inhaled during

breathing, leading the bacteria directly to the lung where they are phagocyted by alveolar

macrophages (white blood cells). Inside the macrophages, the bacilli reside in a membrane-

bound vacuole, and for this reason some are able to avoid fusion with lysosomes and posterior

digestion [131], ending up co-existing with the macrophages [132]. They multiply and eventually

escape the lung through the bloodstream and lymphatic system, spreading to other organs of

the body, resulting in the extra-pulmonary TB [133]. Moreover, M. tuberculosis may exist within

a granulomas consisting of macrophages and giant cells, T cells, B cells, and fibroblasts, and

these granulomas can prevail not only in the lung, but in other organs as well. In latent

infections, the state of the bacteria within the granuloma is unknown.

Despite 100 years of research, infection with M. tuberculosis is still a major health

problem worldwide and one of the main causes of human death by a particular infectious agent.

Inappropriate monotherapy and intermittent treatment are the main causes of drug resistance,

and facilitate the selection and transmission of resistant strains within communities. In 2012,

there were 8.7 million new cases of TB globally, with 1.4 million deaths in the same year, which

confirms TB as a serious global health issue [128]. Of the 8.7 million incident TB cases in 2012,

1.1 million (13%) were among people living with HIV [128].

Currently available chemotherapy includes first-line drugs such as isoniazid (INH),

rifampicin (RIF), ethambutol (EMB) and pyrazinamide (PYZ) [134]. These drugs are

administered orally and are shown to have excellent potency against M. tuberculosis. Isoniazid,

a prodrug activated by the catalase peroxidase (KatG) enzyme, is the most well-known and

used drug to treat TB [135]. Activated isoniazid inhibits NADH-dependent enoyl-ACP reductase,

which plays a role in the synthesis and elongation of mycolic acids on the cell wall [135].

Rifampicin binds to the β-subunit of the RNA polymerase, preventing the formation of the

messenger RNA chain [136]. Pyrazinamide is a prodrug converted into pyrazinoic acid by the

pyrazinamidase enzyme [135]. The protonated pyrazinoic acid accumulates in the cell and

causes cytoplasmic acidification and reduces cell membrane energy, disrupting the proton

motive force and affecting membrane transport [136]. Ethambutol is a bacteriostatic agent that

inhibits the polymerization of arabinogalactan, consequently preventing its biogenesis formation

on the cell wall [137].

25

The second-line drugs used for the treatment of TB are aminoglycosides (e.g. amikacin,

kanamycin), polypeptides (e.g. capreomycin, viomycin, enviomycin), fluroquinolones (e.g.

ciprofloxacin, levofloxacin, moxifloxacin), thioamides (e.g. ethionamide, prothioamide,

cycloserine) [134]. They are used as alternative agents to treat TB when the bacilli are resistant

to first-line drugs and they are less effective, more toxic, and unavailable in many countries due

to high costs [138].

The effect of anti-TB drugs resistance has been around since the first anti-TB agent,

streptomycin, appeared on the market as a monotherapy. Multi-drug resistant (MDR) TB is

defined by resistance to, at least, rifampicin and isoniazid, whereas extensively drug-resistant

(XDR) TB is defined as MDR-TB with additional resistance to any fluoroquinolone and to, at

least, one of the injectable drugs (capreomycin, kanamycin and amikacin). Drug resistance in

TB is the result of spontaneous mutation in combination with selection by poor programmatic

and individual care performance [139]. Globally, WHO estimates that 3.7% of new tuberculosis

cases and 20% of re-treatment cases have MDR-TB [128]. By the end of 2012, 84 countries

had reported at least one case of extensively drug resistant strains (XDR-TB) [128].

M. tuberculosis is intrinsically resistant to numerous antibiotics, and only a few drugs are

effective for treatment [140]. This intrinsic resistance is partially due to the thick, lipid-rich cell

wall, which is an important characteristic of mycobacteria, limiting the penetration of antibiotics.

The cell wall consists of a layer of peptidoglycan surrounding the cell’s basic lipid bilayer. A

second layer of arabinogalactin runs parallel to the peptidoglycan layer, surrounding it in an

intricate, sugary shell. A third layer extends perpendicular to the arabinogalactin shell and

consists of a complex network of mycolic acids. These long and “sticky” mycolic acids are tightly

bound in a final exterior layer, rendering the bacilli virtually impermeable and almost entirely

waterproof. This complex armor allows M. tuberculosis to be resistant to many antibiotics, avoid

death by acidic and alkaline compounds, and prevent cellular phagolysosomal fusion, allowing it

to successfully evade lysis. Figure 8 depicts a simple visual representation of M. tuberculosis

cell wall. Moreover, mycobacteria possess various defence systems against the activity of

antibiotics, including potent β-lactamases and other drug-neutralizing enzymes [140]. Unlike

many other bacteria, M. tuberculosis harbors no resistance plasmids, and drug resistance

arises through the acquisition of specific chromosomal mutations [141].

The current short-course treatment guideline aims for a complete elimination of active

and dormant bacilli involves two phases. During the initial phase four drugs (usually isoniazid,

rifampicin, pyrazinamide and ethambutol) are given daily for two months. The continuation

phase in which fewer drugs (usually isoniazid and rifampicin) are administered for an additional

4 months, either daily or 3 times per week, targets the killing of any remaining or dormant bacilli

in order to prevent relapse [128].

26

Figure 8 – Schematic structure of M. tuberculosis cell wall and action site of first-line anti-TB drugs.

Intercalation of hydrophilic arabinogalactan and hydrophobic mycolate containing layers creates an

extremely impermeable envelope for antibiotic penetration. Small molecules and nutrients are transported

through porin channels that are deposited through these layers. Adapted from [140].

The search for new anti-TB drugs is an important key in this fight, but searching for new

drug delivery strategies may also play an important role. Alternative delivery systems, such as

nanocarriers for anti-TB drugs, may reduce administration frequency and shorten periods of

treatment, hence improving patient compliance and efficacy of treatment, and reduce drug

related toxicity. For example, isoniazid, rifampicin and pyrazinamide are limited by

hepatotoxicity [142], a side effect due to the action of the drug on hepatocytes rather than

macrophages, the primary host cells that harbor M. tuberculosis. Thus, a delivery mechanism

that introduces these antibiotics selectively into macrophages would greatly increase their

therapeutic efficacy by achieving higher concentrations of the antibiotics in site of infection

without exposing the patient to high systemic concentrations that cause toxicities. Because M.

tuberculosis resides and multiplies within host mononuclear phagocytes and because

mononuclear phagocytes internalize particles more efficiently than other host cells,

encapsulation of anti-TB drugs in nanoparticles offers a mechanism for specific targeting of M.

tuberculosis-infected cells. Indeed, because nanoparticles have been shown to be taken up by

macrophages of the reticuloendothelial system and to accumulate in the liver, spleen and lung

[143–145], they are ideally suited to treat M. tuberculosis, which infects macrophages in these

organs. As macrophages exhibit a number of receptors, it is also possible to modify the surface

of these nanocarriers to achieve active targeting of macrophages. Sugars, such as mannose

27

[146] and lactose [147], are among the most commonly used for this purpose, since these

receptors are highly expressed in macrophages. Particle size is also an important characteristic

in passive targeting of macrophages, since they affect the success of internalization within

these cells. In this regard, particles with diameters of about 500 nm have been reported as ideal

to undergo phagocytosis by alveolar macrophages [148].

A variety of different nanocarriers have been produced and tested in vitro and in vivo as

delivery platforms for anti-TB drugs. Table 2 summarizes major data on anti-TB nanoparticulate

formulations.

Table 2 - Dosage forms of TB nanomedicine. From [149].

Mode of delivery and

dosage forms Drug evaluated Animal model

Sustained release

(days)

Plasma Organs

Oral

PLG NPs

Lectin-PLG NPs

Alginate NPs

SLNs

Inhalable

PLG NPs, nebulized

PLG NPs, insufflated

Lectin-PLG NPs

Alginate NPs

SLNs

Injectable

PLG NPs

RIF, INH, PZA

EMB

Streptomycin

Ethionamide

Econazole

Moxifloxacin

RIF, INH, PZA

RIF, INH, PZA, EMB

Econazole

RIF, INH, PZA

RIF, INH, PZA

RIF

RIF, INH, PZA

RIF, INH, PZA, EMB

RIF, INH, PZA

RIF, INH, PZA

Mice, guinea pigs

Mice

Mice

Mice

Mice

Mice

Guinea pigs

Mice, guinea pigs

Mice

Mice

Guinea pigs

Guinea pigs

Guinea pigs

Guinea pigs

Guinea pigs

Mice

6−8

3

4

6

5

4

6−14

7−11

6

8

6−8

0.25

6−14

7−11

5

32

9−11

7

7

5−7

6

6

15

15

8

10

9−11

0.3

15

15

7

36

28

2. Materials and Methods

2.1. Materials

2.1.1. Lipids

All lipids used in this work are well tolerated for human use and recognized as safe

(GRAS status) by FDA.

2.1.2. Surfactant

In this work was used a nonionic surfactant. Surfactants (also known as surface-active

agents or emulsifiers) are essential to stabilize lipid nanoparticles dispersions and prevent

particle agglomeration. Surfactants are amphipathic molecules that possess a hydrophilic

moiety (polar) and a lipophilic moiety (non-polar), which together form the typical head and the

tail of surfactants. At low concentrations, surfactants adsorb onto the surface of a system or

interface. They reduce the surface or interfacial free energy and consequently reduce the

surface or interfacial tension between the two phases [153]. The relative and effective

proportions of these two moieties are reflected in Hydrophilic-Lipophilic Balance, or HLB value.

The HLB scale, defined by Griffin, ranges in value from 1 to 20. Relative hydrophilicity increases

with increasing HLB value. A value of 1 represents a strongly hydrophobic surfactant.

Surfactants with HLB values in the range of 8–18 are suitable for the preparation of lipid

nanoparticles through oil-in-water dispersion medium [154].

The surfactants used include ionic, nonionic and amphoteric surfactants. Ionic surfactants

are traditionally thought to infer electrostatic stability, whilst nonionic surfactants are traditionally

thought to infer steric repulsion stability [153]. Nonionic surfactants are preferred for oral and

parenteral preparations over those with ionic properties because of their lower toxicity and

irritancy. In general, the toxicity of surfactants decreases in the order cationic > anionic >

nonionic > amphoteric [154].

29

2.1.2.1. Hexadecane

Hexadecane 99% was purchased from Sigma-Aldrich, and it was used as hydrophobic

agent for osmotic stabilization of the lipid nanoparticles. Hexadecane, also called cetane, is a

straight chain, saturated alkane hydrocarbon with molecular formula C16H34 and molecular

weight 226.4 g/mol. Its melting point is 18ºC, thus is a liquid at room temperature with a density

of 0.773 g/ml. Its research applications include the preparation of emulsions in water [157]. Its

structure is shown in Figure 9.

Figure 9- Structural formula of hexadecane

2.1.3. Bioactive Compounds

2.1.3.1. Rifampicin

Rifampicin is a semisynthetic antibiotic derivative of rifamycin B and it was purchased

from Sigma-Aldrich. Rifampicin is an orange-brown to red-brown crystalline powder very slightly

soluble in water at neutral pH (2.5 mg/ml, 25ºC) [158]. It is soluble in dimethylsulfoxide

(~100mg/mL), dimethylformamide, methanol (16 mg/ml, 25ºC), chloroform (349 mg/ml, 25°C),

ethyl acetate (108 mg/ml, 25°C), and acetone (14 mg/ml, 25°C) [158]. Its molecular weight is

822.95 g/mol and its chemical formula is C43H58N4O12 [158]. Its structure is shown in Figure 10.

Figure 10- Structural formula of rifampicin

30

2.1.3.2. Pyrazinamide

Pyrazinamide, the pyrazine analogue of nicotinamide, is a white, crystalline powder,

stable at room temperature and it was purchased from Sigma-Aldrich. Pyrazinamide is also

known as pyrazinecarboxamide and its molecular weight is 123.11 g/mol [159]. Its chemical

formula is C5H5N3O and its structural formula is shown in Figure 11 [159]. It is soluble in water

(15 mg/mL) and chloroform, slightly soluble in ethanol (95%) and very slightly soluble in polar

organic solvents [159]. Pyrazinamide is used to form polymeric copper complexes, create

pyrazine carboxamide scaffolds useful as a component of mycobacteria identification kits.

Figure 11 - Structural formula of pyrazinamide

2.1.3.3. β-carotene

For this work, β-carotene was also selected as model lipophile. β-carotene delivery is of

interest in the food industry, the nutraceutical industry, the skin care industry, and marketers of

antioxidant medications. β-carotene possesses a molecular weight of 536.87 g/mol and a

molecular formula of C40H56. It is a strongly-coloured red-orange pigment which belongs to a

group of plant compounds called carotenoids. Carotenoids consist of an isoprene polymer

backbone. β-carotene is composed of two retinyl groups, and is broken down in the mucosa of

the small intestine to retinal, which is a form of vitamin A. β-carotene can be stored in the liver

and converted in two molecules of vitamin A, being a provitamin. β-carotene is insoluble in

water, making it an excellent model small molecule lipophile drug. Figure 12 shows its

molecular structure.

Figure 12 – Structural formula of β-carotene

31

2.1.4. Water

The water used in all experiments was purified water by reverse osmosis and was

obtained from a Milli-Q Plus, Millipore system. The water had an electrical conductivity of 0.5-

1.5 μS/cm.

2.2. Methods

2.2.1. Preparation of Lipid Nanoparticles

Lipid nanoparticles were prepared by ultrasonication and simple magnetic stirring

methods. Both aqueous and lipid phases are separately prepared before mixing. The aqueous

phase is composed of water (≥ 80.0% w/w), non-ionic surfactant (≤8.0% w/w) and hexadecane

(≤3.5% w/w). Six formulations of empty SLNs was prepared ranging lipid content: SLN_1

represents the formulation with the lowest lipid phase content and SLN_6 represents the

formulation with the highest lipid phase content. For NLC formulations was used a combination

of natural oil with the MCFA. The content of bioactive compounds incorporated in lipid

nanoparticles are shown at Table 3.

Table 3 - Composition % (w/w) of bioactive compounds loaded to lipid nanoparticles.

In detail, the lipids are melted in a warm water bath above their melting points. Different

temperatures conditions up than 50ºC were tested. When the MCFA or the MCFA/natural oil

combination was fully melt, a hot aqueous solution with nonionic surfactant and hexadecane

Formulation β RIF PYZ

2RIF_SLN - 0.06 -

2RIF_NLC - 0.06 -

4RIF_SLN - 0.11 -

4RIF_NLC - 0.11 -

7RIF_SLN - 0.20 -

7RIF_NLC - 0.20 -

4PYZ_SLN - - 0.11

4PYZ_NLC - - 0.11

β10-SLN 0.3 - -

β10-NLC 0.3 - -

32

preheated at the same conditions was added. A hot coarse o/w was obtained under magnetic

stirring at 750 rpm for 1min and was subjected to sonication using a probe (MS72) sonicator

(Bandelin, Germany) during 5 minutes. Hot o/w emulsion was allowed to cool to room

temperature under magnetic stirring at 750 rpm to obtain the lipid nanoparticles. After 2h, the

samples of each formulation were stored both in a refrigerator at 4ºC and at room temperature.

For active agents-loaded lipid nanoparticles, the active agent was added to the lipid phase, 15

minutes earlier the addition of aqueous solution. Figure 13 shows the procedures.

Figure 13 – Lipid nanoparticles production. Left: Lipid phase and aqueous phase in the warm water bath

under same conditions (temperature and agitation). Top Right: Lipid phase (on the right) and aqueous

phase (on the left) after weight. Middle Right: Addition of hot aqueous phase in melt lipid phase. Bottom

Right: Sonication of hot pre-emulsion.

33

2.2.1.1. Lyophilisation of Lipid Nanoparticles

Lyophilisation (or freeze-drying) was the final step of the production process. This

process promotes water removal from a frozen sample, using sublimation and desorption under

vacuum. Many pharmaceutical products, mainly heat sensitive compounds, are dried using this

technique, since it improves the long-term physic-chemical stability and prevents degradation

reactions such as hydrolysis. Lyophilisation is also used with nanoparticles to prevent particle

aggregation.

The aqueous samples of lipid nanoparticles were fast frozen under −80 °C in a deep

freeze for 5h in ultra-low refrigerator. Then, the samples were moved to the freeze-drier (Christ

Alpha 1-2 LD) during 48h in order to get powder of lipid nanoparticles.

2.2.2. Characterization of Lipid Nanoparticles

2.2.2.1. Particle Size

In this work, a Zetasizer Nano ZS, Malvern Instruments (Malvern, UK) was used to

measure the size of lipid nanoparticles produced. Particle size measurements were made in

disposable cells at 25°C, by non-invasive back scatter, with dynamic light scattering detected at

an angle of 173°. One ml of samples were used and 1:10 dilutions were performed (900μL

ultrapure Milli-Q water + 100μL sample), to avoid multiple scattering phenomenon. The effective

hydrodynamic diameter was calculated from the diffusion coefficient by the Stokes-Einstein

equation (Eq.1) using the method of cumulants as an alternative to distribution analysis. In

summary, the cumulants analysis gives a good description of the size that is comparable with

other methods of analysis for spherical, reasonably narrow monomodal samples, with PDI value

below 0.1. For samples with a slightly increased width, the Z-average size and PDI will give

values that can be used for comparative purposes. All measurements were performed in

triplicates with 20 runs by measurement.

2.2.2.2. Zeta Potential

Zeta potential measurements were also performed using a Malvern Zetasizer Nano ZS,

Malvern Instruments (Malvern, UK) at 25ºC. The samples were diluted 1:10 in ultrapure Milli-Q

water and placed at a folded capillary cell (DTS1060) where an alternating voltage of ±150mV

was applied. The measured electrophoretic mobilities were converted into zeta potential values

using the Smoluchowski approximation. All measurements are performed in triplicate with 20

runs per measurement.

34

2.2.2.3. Particle Morphology

Morphological observations of the SLNs and NLCs were performed using a transmission

electron microscope (TEM). In this work, 20 µL of samples of with lipid nanoparticles previous

analysed by DLS was deposited over a carbon covered TEM copper grid and then dried at room

temperature. TEM images were collected using a microscope H-8100 Hitachi, operated with

200 kV of acceleration voltage incorporated with a CCD MegaView II bottom-mounted camera.

2.2.2.4. Encapsulation Efficiency

An indirect method was used to determine the encapsulation efficiency of lipid

nanoparticles using Eq.3 as explained in chapter 1.

For all three active compounds loaded within lipid nanoparticles the same following process

was used. After production, lipid nanoparticles were diluted and washed in milli-Q water. The

dispersion was vortexed and then centrifuged at 12000 rpm for 30 min at 4ºC using centrifuge

(Centrifuge 5810 R, Eppendorf). The supernatant was collected and the pellet was resuspended

in fresh milli-Q water. This procedure was repeated three times. After washes, the amount of

active agents in the supernatant was determined from its absorption using a double beam UV–

VIS spectrophotometer (Model U-2000, Hitachi) and 1cm quartz cells.

The amount of drug dissolved in supernatant can be quantified by spectrophotometer,

since absorbance of a solution at a specific wavelength is proportional to the solute's

concentration, as is expressed by Beer-Lambert's law:

A= εlC Eq.4

where ε is the molar absorptivity (a constant that indicates how well the solute absorbs light of a

particular wavelength, in units of M-1cm-1), l is the solution’s length the beam has to cross (1.0

cm for the quartz cell), and C is the solute’s concentration (mol/L).

In this work, the measures of absorbance were done at 333 nm for rifampicin and at 548

nm for β-carotene. Pyrazinamide was analysed based on the formation of a coloured complex

between the drug and sodium nitroprusside at alkaline pH with a mixing ratios of 4:1, which

could be measured at 495nm (Figure 14). The solution of sodium nitroprusside at alkaline pH

was prepared mixing a solution of 2% sodium nitroprusside in milli-Q water with a 2N sodium

hydroxide solution in a ratio of 1:1 [160].

35

Figure 14 – Pyrazinamide standard solutions used in constructing of the calibration curve, after reacting

with alkaline sodium nitroprusside. Pyrazinamide concentration increases from left to right.

In order to calculate the percentage of active compound incorporated is required to do a

calibration curve by preparing several samples with well-known concentrations and measuring

theirs corresponding absorbance at a particular wavelength. The resulted equation is then used

to determine the mass concentration of the sample, through measuring the absorbance at same

wavelength. Calibration curves of rifampicin, pyrazinamide and β-carotene were obtained by

measuring solutions with known concentrations of the active compounds dissolved in milli-Q

water. Samples were estimated in triplicates. Resulting calibration curves and respective

equations are represented in Figure 15-17.

Figure 15 - Rifampicin calibration curve

36

Figure 16 - β-carotene calibration curve

Figure 17 - Pyrazinamide calibration curve

37

3. Results and Discussion

3.1. Study of Fabrication Parameters

3.1.1. Empty SLN

The first objective of this work was to study SLNs at room temperature and their

production conditions by miniemulsion using ultrasonication, based on a MCFA as solid lipid.

The greatest advantage of this procedure is the easily production of SLNs without using organic

solvents. In this work, a nonionic surfactant was employed, providing a steric hindrance,

avoiding emulsion droplets from coming close to each other and thus preventing flocculation

and coalescence. In this process, the MCFA is heated above its melting point. Aqueous solution

of surfactant is mixed with lipid melt at the same temperature. Shear force from sonication will

facilitate the formation of miniemulsion. The droplet size is governed by energy dissipation,

temperature, surfactant and lipid concentration and other factors. The hot emulsion is then

cooled down to form solid particles.

Since the purpose of SLNs produced are for oral administration, the particles must be an

average size less than 400 nm to easily cross intestinal cells [59]. Furthermore, the fabrication

of particles comprises 100-200 nm it would be better, since it was reported particles smaller

than 200 nm usually remain invisible to the reticulo-endothelial system (RES) and keep on

circulation system over a prolonged period of time [35]. For this purpose, it was tested the

influence of different parameters, such as applied lipid amount, temperature and sonication step

on mean particle size expressed as Z-average mean size (Z-ave), polydispersity index (PDI)

and zeta potential (ZP) (Table 4, Figure 18 and Figure 19) .

Mean particle size of all formulations ranged from 69 ± 5 nm to 601 ± 100 nm and PDI

from 0.87 ± 0.09 to 0.11 ± 0.02 (Table 4 and Figure 18). Since all the formulations shows a

PDI higher than 0.1, this indicates that none are monodisperse. Finally, the ZP range from -35.8

± 0.8 mV to -22.7 ± 0.7 mV (Table 4 and Figure 19), indicating good physical stability since

particles aggregation was not likely to occur due to the electrostatic repulsion between the

particles. For long-term stability a balance between electrostatic repulsion and steric effect of

surfactant must be obtained.

.

38

Table 4 - Physicochemical characteristics of empty SLN at different conditions (mean ± SD, n = 6).

Non-sonicated Sonicated

T (ºC)

Formulation Z-Ave (d.nm)

PdI ZP (mV) Z-Ave (d.nm)

PdI ZP (mV)

a

SLN_1 601 ± 100 0.70 ± 0.08 -29.6 ± 0.6 146 ± 2 0.18 ± 0.05 -25.8 ± 0.7

SLN_2 250 ± 6 0.56 ± 0.03 -27.5 ± 0.2 130 ± 9 0.18 ± 0.02 -27.8 ± 1.5

SLN_3 224 ± 28 0.46 ± 0.11 -28.8 ± 0.1 105 ± 1 0.18 ± 0.02 -27.8 ± 0.8

SLN_4 284 ± 19 0.87 ± 0.09 -30.4 ± 0.8 115 ± 1 0.18 ± 0.04 -29.4 ± 0.7

SLN_5 249 ± 16 0.70 ± 0.07 -29.3 ± 0.3 121 ± 3 0.15 ± 0.02 -28.6 ± 0.6

SLN_6 326 ± 6 0.85 ± 0.09 -32.4 ± 0.3 121 ± 1 0.18 ± 0.05 -28.1 ± 1.2

b

SLN_1 299 ± 8 0.55 ± 0.06 -31.0 ± 0.4 139 ± 2 0.17 ± 0.03 -27.2 ± 2.0

SLN_2 262 ± 13 0.44 ± 0.07 -29.9 ± 0.5 134 ± 23 0.18 ± 0.02 -25.1 ± 1.0

SLN_3 231 ± 25 0.54 ± 0.16 -28.3 ± 0.6 121 ± 25 0.20 ± 0.04 -30.3 ± 0.7

SLN_4 255 ± 8 0.47 ± 0.09 -30.0 ± 0.8 122 ± 10 0.17 ± 0.04 -28.6 ± 0.6

SLN_5 411 ± 22 0.51 ± 0.15 -26.5 ± 0.2 122 ± 11 0.19 ± 0.02 -27.2 ± 0.9

SLN_6 330 ± 55 0.45 ± 0.35 -35.8 ± 0.8 109 ± 19 0.18 ± 0.04 -31.7 ± 0.9

c

SLN_1 515 ± 140 0.52 ± 0.13 -30.6 ± 0.6 141 ± 1 0.14± 0.03 -28.4 ± 0.8

SLN_2 155 ± 4 0.26 ± 0.01 -28.3 ± 0.7 126 ± 7 0.15± 0.02 -26.8 ± 0.9

SLN_3 140 ± 1 0.20 ± 0.01 -29.3 ± 0.6 97 ± 30 0.23 ± 0.09 -28.9 ± 1.0

SLN_4 196 ± 2 0.30 ± 0.03 -31.5 ± 0.2 119 ± 1 0.13 ± 0.02 -29.2 ± 0.6

SLN_5 149 ± 1 0.29 ± 0.02 -29.9 ± 0.5 120 ± 1 0.17 ± 0.05 -28.5 ± 3.9

SLN_6 179 ± 1 0.25 ± 0.01 -25.5 ± 0.7 122 ± 2 0.18 ± 0.01 -28.2 ± 0.7

d

SLN_1 333 ± 50 0.41 ± 0.03 -30.0 ± 0.5 139 ± 1 0.11 ± 0.02 -26.6 ± 0.6

SLN_2 93 ± 7 0.30 ± 0.08 -29.1 ± 0.6 87 ± 12 0.21 ± 0.02 -22.7 ± 0.7

SLN_3 144 ± 44 0.87 ± 0.05 -12.3 ± 0.6 105 ± 1 0.23 ± 0.03 -28.4 ± 0.6

SLN_4 90 ± 3 0.46 ± 0.03 -29.3 ± 0.3 69 ± 5 0.19 ± 0.03 -29.4 ± 0.7

SLN_5 99 ± 2 0.49 ± 0.05 -34.7± 1.5 97 ± 30 0.23 ± 0.09 -28.9 ± 1.0

SLN_6 121 ± 2 0.40 ± 0.02 -30.6 ± 0.3 126 ± 7 0.19 ± 0.03 -26.0 ± 0.2

39

Figure 18- Z-ave and PDI of empty SLNs with different content of MCFA (SLN_1 represents the lowest

lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest

temperature (a) to highest temperature (d).

40

Figure 19 - Zeta Potential of empty SLNs with different content of MCFA (SLN_1 represents the lowest

lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest

temperature (a) to highest temperature (d).

41

3.1.1.1. Influence of Temperature

The effect of temperature on pre-emulsion was investigated. Four different temperatures

up than 50ºC were applied. The main influence of temperature was observed on non-sonicated

samples. Table 4 shows a notorious size reduction of non-sonicated particles with the increase

of temperature, while PDI and ZP values remain with non-significant variation. These results are

expected since in general, the increase of temperature usually decreases the viscosity of the

lipid and aqueous phases and hence the efficiency of the agitation and mixture is increased. In

other words, increasing the fusion temperature of the lipids results in smaller droplet/particle

sizes [113].

3.1.1.2. Influence of Lipid Content

The influence of MCFA content on SLNs properties were also investigated (Table 4 and

Figure 18-19). From analysis of non-sonicated samples in a, c and d of Figure 18, it seems

evident that particle size of SLNs prepared with the lowest concentration of MCFA are larger

when compared with other SLNs. In addition, PDI of these samples are also high. However, ZP

appears not be affected as well as all features considered of lipid particles obtained by

sonication.

As PDI of non-sonicated SLNs produced with lowest lipid content are also very high, the

Ostwald ripening effects could be an explanation for their larger sizes. Ostwald ripening is a

thermodynamically driven process, in which smaller particles dissolve and redeposit onto the

surface of larger particles. This process occurs because smaller particles have larger surface

area and higher surface energy and hence higher Gibbs free energy than the larger particles.

All systems tend to attain lowest Gibbs free energy. In other words, larger particles are more

energetically stable and thus, preferred over smaller particles. Ostwald ripening can be reduced

by minimizing PDI in the particle size but it cannot be prevented.

In conclusion, although in general low lipid content decreases the aggregation

phenomenon, it may also increase the Ostwald ripening effects and causes the migration of

surfactant molecules from the particle interface to aqueous medium decreasing the electrostatic

repulsive force and leading particle aggregation.

42

3.1.1.3. Influence of Sonication

The influence of sonication on SLNs features was represented in Table 4 and Figures

18-19.

Figure 20 - Macroscopic aspect of empty SLNs: (left) - non-sonicated sample; (right) –

sonicated sample

Comparing the macroscopic aspect between non-sonicated and sonicated SLNs

suspension (Figure 20), it is possible to see that non-sonicated SLNs dispersion has a milky

aspect, while sonicated SLNs dispersion are more clear and bluish. From literature, usually

colloidal dispersions with droplet sizes between 20–100 nm are transparent or bluish, while

colloidal dispersions with larger droplets sizes up to 500 nm has a milky aspect [107]. Thus,

through macroscopic analysis, it can be expected a decreased on mean SLNs particle size of

sonicated samples.

The particle size reduction is confirmed by DLS results. At Figure 18, it is clear the effect

of sonication in both Z-ave and PDI of MCFA SLNs. All sonicated SLNs show a mean Z-ave

lower than 150 nm. This is due to shear stress, which is resultant from the application of

ultrasound energy that promotes a shear on MCFA molecules, impairing aggregation, thus,

avoiding the formation of larger particles. Despite of this significant effect on particle size,

ultrasonication does not appear to play an essential role in physical stability of SLNs, since ZP

is very similar between non-sonicated and sonicated SLNs, although there is an insignificant

decrease in absolute ZP value (Figure 19).

In conclusion, the application of ultrasound energy seems to decrease the mean SLNs

particle size and also homogenize the SLNs in suspension, since PDI values decrease when

sonication is applied.

43

3.1.1.4. Lyophilisation

Figure 21 – Powder resulting of SLN_1b_II lyophilisation

Lyophilisation was applied to some SLNs samples in order to remove the excess water

and also to enhance stability of powder SLNs, since Ostwald ripening can be avoided and

particles aggregation decreased. The obtained powder of SLN_1b_II is shown at Figure 21.

Table 5 presents the Z-ave, PDI and ZP of the SLN_1b_II before and after lyophilisation.

Z-ave of lyophilised SLNs is slightly larger when compared to liquid dispersion, increasing from

153 ± 31 nm to 191 ± 4 nm. However, PDI of lyophilised SLNs increased from 0.13 ± 0.04 to

0.49 ± 0.10, resulting on a sample far polydisperse. Examining particle size distribution of

SLN_1b_II before and after lyophilisation (Figure 22), it is possible to realize the appearance of

a second particles population larger than 1 µm in reconstituted lyophilised sample. This larger

population could be result of aggregation phenomenon, indicating physical instability of SLNs

and explaining the increase of PDI. However, contrary as expected, absolute ZP value

increased from 30 ± 2 mV to 35 ± 2 mV, meaning an increase on particles stability. An

explanation for these results could be an insufficient stirring force/time applied on resuspension

of lyophilised SLNs in milli-Q water. If the attractive forces holding the aggregate together are

very large compared with the typical forces used in stirring, mixing or ultrasonic probes, the

system is regarded as permanently aggregated. Thus, the larger population of SLNs on

lyophilised SLN_1b_II could be a result of flocculation. To explore this hypothesis, it could be

done a comparative study between different forces used to resuspension of lyophilised SLNs.

In addition, it could be investigated the combination of a cryoprotector previously to

lyophilisation since it was found that cryoprotective agents preserve the physicochemical

properties of SLNs and decrease SLNs aggregation. Typical cryoprotectors are sorbitol,

mannose, trehalose, glucose, and polyvinylpyrrolidone. The best results occurred when

cryoprotector is in concentrations of 10-15% [162].

44

Table 5 – Physicochemical characteristics of the SLN_1b_II. Values are mean ± SD, n = 3

Formulation Z-Average

(nm) PDI

ZP

(mV)

SLN_1b_II 153 ± 32 0.13 ± 0.04 -30 ± 2

Lyophilised SLN_1b_II 191 ± 4 0.49 ± 0.10 -35 ± 2

Figure 22 – Particle Size Distribution by Intensity of SLN_1b_II before and after lyophilisation.

3.1.1.5. Long-Term Stability

In this work, SLNs formulations were stored at room temperature (25ºC) and in the

refrigerator (4ºC), during seven months. The long-term stability of both formulations was

assessed by means of particle size (Z-ave and PDI) and ZP measurements. The stability was

also monitored by macroscopic observations.

The samples stored at room temperature remained with a milky-like appearance,

although some of samples show particle sedimentation. However, during storage for more than

one week some unpredictable gelation occurred in SLNs containing samples stored at 4ºC.

Gels are more structured than liquids. SLNs are able to organize themselves in superstructures.

The change in morphology of lipid nanoparticles from spheres to platelets is responsible for the

gelation of SLN dispersions [163]. Depending on the composition, especially of the emulsifier(s)

and the amount of lipid matrix, a gelation of the normally liquid dispersions can be observed on

storage [163].

Table 6 shows the obtained Z-ave, PDI and ZP results for SLN_4b_I, an example of a

macroscopic milky-like colloidal SLNs dispersion stored at room temperature. From results

analysis, it can notice a decrease in the absolute ZP value from 39.1 ± 0.9 mV to 28.3 ± 0.6 mV,

indicating changes in the particles structure. This can be confirmed by the DLS measurements

where particle size growth was observed (Table 6 and Figure 23). The mean particle size

45

remained lower than 400 nm, although there was an increasing on the size from 154 ± 25 nm to

223 ± 54 nm. Since PDI and particle size remain relatively low, homogeneously sized SLNs

displayed a satisfactory long-term stability.

Table 6 - Physicochemical characteristics of the SLN_4b_I. Values are mean ± SD, n = 3.


(nm) PDI

ZP

(mV)

SLN_4b_I 154 ± 25 0.07 ± 0.03 -39.1 ± 0.9

SLN_4b_I after 7months 223 ± 54 0.05 ± 0.02 -28.3 ± 0.6

Figure 23 - Particle Size Distribution by Intensity of SLN_4b_I after one day and 7 months after

production.

The shape and surface morphology of empty SLNs after seven months were also

analysed by TEM and the resulting images are represented in Figure 24. In the TEM study, the

size of the SLNs was found to be in agreement with the DLS. All the particles were found to be

smoothly spherical or oval in shape with a well-defined periphery. No obvious aggregation of the

SLNs was observed. It should be noted, however, that the particles align in three dimensions,

whereas the micrographs only allow particle presentation in two dimensions so that the given

particle dimensions are only estimates due to the fact that a particle observation exactly top-on

and edge-on, respectively, is rare and cannot be discriminated from slight particle twists, in

addition.

46

Figure 24 – TEM image of SLN_4b_I after 7 months.

3.1.2. Empty NLC

For NLC production, the fabrication procedure was similar to the SLNs fabrication and

was described in chapter 2.

Table 7 presents the Z-ave, PDI and ZP of the NLC. The Z-ave of NLC is larger when

compared to SLN_4b. Since both lipid nanoparticles have an equal percentage of lipid content

and were produced at same conditions, the increase of the size from 119 ± 0 nm to 282 ± 56 nm

are related with the use of natural oil. The combination of different chain fatty acid triglycerides

promotes changes in structure of lipid nanoparticles. These changes on particles surface are

also supported by the slightly reduction of absolute ZP value from -29.2 ± 0.6 mV to -26.4 ± 0.7

mV. In conclusion, despite the growth of particle size, NLC size remains lower than 400 nm, as

required to easily cross intestinal cells.

Table 7 – Physicochemical characteristics of the empty NLC. Values are mean ± SD, n = 3.


(nm) PDI

ZP

(mV)

NLC 282 ± 56 0.43 ± 0.09 -26.4 ± 0.7

47

3.2. Bioactive Compounds Loaded SLNs and NLCs

The second main goal of this project was to investigate the entrapment efficiency of some

bioactive compounds in MCFA based lipid nanoparticles. The anti-TB drugs rifampicin and

pyrazinamide were chosen to encapsulate within lipid nanoparticles and β-carotene was also

used as lipophilic compound model.

From the first part of this study, it had to be considered a compromise between the

adequate size, a low PDI and also a good stability of the samples for the final choice of the

parameters to be used in loaded lipid nanoparticles production. As mentioned above, the

desired formulations have to be lower than 400 nm. Furthermore, the fabrication of particles

comprises 100-200 nm it would be better, since it was reported particles smaller than 200 nm

usually remain invisible to the reticulo-endothelial system (RES) and keep on circulation system

over a prolonged period of time [35]. Taking all this considerations into account, the final

parameters chosen to lipid nanoparticles production were those that correspond to the SLN_4

formulation and the temperature of lipid melting must be up 60ºC, followed by 5 minutes of

sonication. Optimal temperature also depends of drug characteristics.

Figure 25 shows the difference in macroscopic appearance of empty and loaded NLCs

before and after sonication. After sonication, all the dispersions are significantly more

transparent.

Figure 25 – Differences in macroscopic appearance of empty and loaded NLCs immediately before and

after sonication.

48

3.2.1. β-carotene

β-carotene was incorporated in SLNs and NLCs. The Z-ave, PDI and ZP of loaded

particles were measured one day after production and the results are represented in Table 8.

The %EE of the prepared lipid nanoparticles were determined indirectly, by calculating the

amount of free β-carotene (non-encapsulated) present in the aqueous phase of dispersions, as

explained in chapter 2 and was also represented in Table 8. As expected, SLNs and NLCs

loaded with β-carotene are larger when compared with empty SLNs (Table 4) and empty NLCs

(Table 7). Particle sizes remain lower than 400 nm, as required. No important changes seem to

occur on stability of both loaded SLNs and NLCs, since ZP values remain similar from empty

SLNs and NLCs. In addition, comparing with rifampicin (Table 9) and pyrazinamide (Table 10)

loaded-particles, β-carotene shows the highest percentage of encapsulation efficiency. This

result was predictable, since β-carotene is the most lipophilic of these three active compounds.

Also, since β-carotene is less soluble in oil then in fat, the %EE of β10-SLN is higher than β10-

NLC.

Table 8 - Physicochemical characteristics of the β-carotene lipid nanoparticles. Values are mean ±

SD, n = 3.


(nm) PDI

ZP

(mV)

EE

(%)

β10-SLN 281 ± 19 0.34 ± 0.03 -29 ± 5 95 ± 5

β10-NLC 328 ± 65 0.28 ± 0.06 -25 ± 3 83 ± 6

Morphological analysis of β-NLC was performed by TEM and the resulting images are

represented in Figure 26. The size of the particles was in agreement with DLS results. TEM

revealed nanoparticles with almost spherical shapes and a roughness surface. However, it was

also reported the presence of some aggregates and some particles with an irregular shape.

Figure 26- TEM image of β-carotene loaded NLC.

49

3.2.2. Rifampicin

The Z-ave, PDI, ZP and %EE of RIF loaded lipid nanoparticles are presented in Table 9.

As expected RIF-NLCs samples are larger than RIF-SLNs. In addition, RIF loaded particles

show a reduction in the electrical charge at the surface to mean values below -20 mV. These

results could be taken as an indication that RIF is entrapped in the lipid matrix of lipid

nanoparticles. Due to the high solubility of RIF in the lipid core, the encapsulation efficiency was

good enough for all the formulations, ranging between 67 ± 7% and 85 ± 5%. In literature, it was

found that NLCs can encapsulate slightly more amount of drug than SLNs. However, none

significant differences in %EE it seems to exist between RIF-SLNs and RIF-NLCs. Muller et al.

also reported that the presence of liquid lipid in the solid matrix avoids the drug expulsion during

storage that can occur when the lipid matrix undergoes polymorphic transformations from

unstable to more stable configurations [28]. In this work, no long-term stability study was done

to verify this event. And, since, it is obvious that polymorphic crystal transformation during

storage can affect the encapsulation efficiency and the drug release profile of lipid

nanoparticles, additional thermal and crystallization studies must be done in order to a better

control of encapsulation efficiency and the drug release.

Table 9 - Physicochemical characteristics of the rifampicin lipid nanoparticles. Values are mean ±

SD, n = 3.


(nm) PDI

ZP

(mV)

EE

(%)

2RIF_SLN 341 ± 78 0.45 ± 0.12 -20.3 ± 2.2 74 ± 10

2RIF_NLC 391 ± 210 0.62 ± 0.10 -18.5 ± 1.3 67 ± 7

4RIF_SLN 84 ± 1 0.23 ± 0.01 -17.8 ± 0.7 69 ± 1

4RIF_NLC 180 ± 90 0.36 ± 0.08 -19.5 ± 0.9 69 ± 1

7RIF_SLN 181 ± 31 0.34 ± 0.03 -19.2 ± 1.3 79 ± 7

7RIF_NLC 215 ± 5 0.28 ± 0.03 -18.8 ± 0.6 85 ± 5

From TEM analysis represented at Figure 27, it can be observed that RIF-NLCs also

exhibit spherical shapes, which indicates drug loading is not lead to morphological changes.

Again, the particle size was in concordance with particle size analysis data obtained from

dynamic light scattering

50

Figure 27 - TEM image of rifampicin loaded NLC.

In literature, it was found two studies involving the encapsulation of RIF in lipid

nanoparticles. In first one, RIF was loaded into Compritol ATO 888 SLNs fabricated by a

modified microemulsion technique. The particles sizes obtained were 141 ± 13nm, ZP value

was −3.5 ± 0.8mV and %EE was 65 ± 3% [164]. In second one, RIF was encapsulated in group

with pyrazinamide and isoniazid in a stearic acid SLNs produced by emulsion solvent diffusion

with a %EE of 51 ± 5% [89]. Perhaps due to RIF was been encapsulated combined with other

anti-TB drugs the %EE was significant lower than results described in this work. In general,

results obtained in this project seem to be very good, by comparison with reported results by

literature. For one hand, it was obtained higher encapsulation efficiency and for another hand,

fabrication method applied is free-use of organic solvent, meaning less toxicity of the obtained

particles.

3.2.3. Pyrazinamide

The results of Z-ave, PDI, ZP and %EE of pyrazinamide loaded lipid nanoparticles are

represented in Table 10. The %EE value obtained range from 14 ± 8% for PYZ-SLN to 29±15%

for PYZ-NLC. These low results are expected since pyrazinamide is the most hydrophilic

compound in that study, being soluble in water (15 mg/ml). Thus, pyrazinamide partitioned

between the melted lipid and aqueous phase.

However, in literature pyrazinamide was encapsulated in a stearic acid SLNs combined

with isoniazid and rifampicin and %EE reported was 41 ± 7% [89]. Since pyrazinamide is one of

51

anti-TB drugs administered in current chemotherapy, in further studies could be investigated the

combine incorporation of pyrazinamide with other anti-TB drugs such as rifampicin, isoniazid or

ethambutol. Furthermore, the process of cooling from high to room temperature could be

optimized in order to control crystallization processes and consequently the encapsulation

efficiency.

Table 10 - Physicochemical characteristics of the pyrazinamide lipid nanoparticles. Values are

mean ± SD, n = 3.


(nm) PDI

ZP

(mV)

EE

(%)

4PYZ-SLN 217 ± 87 0.31 ± 0.08 -21.0 ± 0.7 14 ± 8

4PYZ-NLC 313 ± 214 0.34 ± 0.17 -22.7 ± 2.4 29 ± 15

The resulting images of TEM analysis of PYZ-NLC are represented in Figure 28. The

size of the particles was in agreement with DLS results. However, TEM revealed nanoparticles

with poorly spherical and irregular shapes. It was also reported the presence of a lot of

aggregates, indicating important alterations on PYZ loaded particles structure.

Figure 28 - TEM image of pyrazinamide loaded NLC

52

4. Conclusions and Further Works

In the present work MCFA SLNs and NCLs with a combination of MCFA and natural oil

as lipid phase were successfully prepared by sonication technique. This technique was simple,

reproducible, prepared nanoparticles without the need of organic solvents or any sophisticated

instruments and has the potential to easily scale up for large scale production.

Furthermore, the lipid nanoparticles obtained in the present work are suitable carrier

systems for the incorporation of different bioactive compounds intended for oral administration,

since mean particle size of all formulations ranged from 69 ± 5 nm to 601 ± 100 nm and the

great part of the formulations revealed a particle size less than 400 nm which easily cross

intestinal cells. The presence of a small quantity of bigger particles was not seen as critical,

because the preparations were intended for oral use and not for intravenous administration.

Also, it was obtained a lot of formulations with a particle size less than 200 nm, which is an even

better result since they will remain invisible to the reticulo-endothelial system (RES) and keep

on circulation system over a prolonged period of time. The Zeta Potential obtained was

negatively enough to ensure a good physical stability of the particles, indicating that the

nonionic surfactant used is a good surfactant for lipid nanoparticles produced with this particular

formulation. In general, the formulations show a PDI higher than 0.1, indicating that none are

monodisperse. To obtain formulations with lower PDI values, such that they could be

considered monodisperse, filtration could be done with porous of low diameters, such as 450

nm or even 200 nm. In industry, other methods to produce lipid nanoparticles could be used,

and some of them, like high pressure homogenization, will naturally produce monodisperse

populations of particles.

Morphological studies using TEM images showed spherical to oval SLNs and RIF-NLCs

with well-defined periphery. The particle size was in concordance with particle size analysis

data obtained from dynamic light scattering. However, the β-NLCs and PYZ-NLCs show an

irregular shape and some tendency to aggregate.

Lyophilisation of lipid nanoparticles seems to be a good alternative to remove excess of

water and to enhance stability of the particles during the storage. Also, the obtained powders

can be used to produce classic solid dosage forms, can be redispersed in water or juice prior to

administration, and can also be used for the filling of hard gelatine capsules. However, further

studies must be done to prevent aggregation of lyophilised particles. It could be explored the

use of an additional cryoprotector previously lyophilisation. Furthermore, other alternatives to

lyophilisation could be investigated such spray drying. This method has been used scarcely for

SLNs formulation, although it is cheaper than lyophilisation

Rifampicin, Pyrazinamide and β-carotene were successful loaded at lipid nanoparticles.

Rifampicin encapsulation efficiencies obtained in this work were higher than the values reported

in literature for encapsulation of rifampicin in lipid nanoparticles. Pyrazinamide shows low

encapsulation efficiencies due to its hydrophilicity leading to partition between lipid phase and

53

aqueous phase. However, pyrazinamide could be encapsulated combined with other anti-TB or

could be done a stearic modification in order to improve its entrapment into solid matrix. In

further studies could be explored the encapsulation of isoniazid and ethambutol in produced

lipid nanoparticles. Nevertheless, further analysis such as DSC must be performed to

characterize the state and the degree of crystallinity of lipid dispersions provide guidance to

better control polymorphic transition of lipid nanoparticles, which have profound impact on

increasing loading capacity, encapsulation efficiency, manipulating release pattern and even

improving long term stability of lipid nanoparticles.

Furthermore, the next step on the characterization of produced lipid nanoparticles must

be in vitro releases and antibacterial activity studies. In addition, further studies related with

cytotoxicity and uptake of lipid nanoparticles could be done using Caco-2 cell line since it is

often used as a model to mimic the GI tract conditions.

In conclusion, despite being essential several further studies to ensure the efficacy of

obtained lipid nanoparticles as drug delivery system for oral route, these initial results are very

promising, and so they should encourage us to go farther.

54

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development and characterization of lipid nanoparticles ... · transporte de fármacos, absorção...

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