UNIVERSITY OF DEBRECENMEDICAL AND HEALTH SCIENCE CENTER

FACULTY OF PHARMACY

NANOCARRIER SYSTEMS IN

PULMONARY DRUG ADMINISTRATION

STUDENT: NGUYEN NGOC VAN ANH

THESIS LEADER: PROF. MIKLOS VECSERNYES, PhD

DEBRECEN, 2013

I AGREE THE CONTENT OF THE THESIS

THESIS LEADER

_______________________

Dr. MIKLOS VECSERNYES

Dean of Faculty of Pharmacy

Debrecen, 8th March 2013

CONTENTS

1. Introduction 4

2. Respiratory tissue organization 7

3. Blood- air barriers 8

3.1. Lung surfactant 9

3.2. Epithelial surface fluids 9

3.3. Pulmonary epithelia 10

3.4. The interstitium and basement membrane11

3.5. Vascular endothelium 12

4. Mechanism of deposition of particles in the lung 14

5. Mechanism of respiratory absorption 15

5.1. Transport through cells: Transcytosis 17

5.2. Paracellular transport: Diffusion between cells 17

6. Significance of nanocarrier systems for pulmonary administration 18

6.1. Micelles as nanocarriers in pulmonary delivery 19

6.2. Liposomal pulmonary drug delivery vehicles 20

6.3. Microemulsions in pulmonary administration 22

7. Micro- and nanoparticle delivery systems…………………………………………… 23

7.1. Microspheres as pulmonary drug carriers 23

7.2. Polymeric nanoparticles 23

7.3. Solid lipid nanoparticles 24

7.4. Dendrimer based nanoparticles 25

8. Application of nanocarrier systems in treatment of non respiratory diseases 26

9. Pulmonary Toxicity of Nanocarriers 30

10. Conclusion 31

Reference 32

3

1. Introduction

The respiratory system is an attractive target for drug delivery, it represents a possible door for the

entrance of therapeutic compounds into the body. Pulmonary administration shows potential advantages

over other routes of administration due to:

i. noninvasive administration (this is limited by parenteral route, which may reduce patient

compliance and requires a prolonged or chronic treatment).

ii. avoidance of first-pass metabolism (this is limited by oral route, because the drug metabolizing

enzymes are in much higher concentrations in the gastrointestinal system and the liver than in the

lungs) [1].

iii. direct delivery of drugs to the target site, leading to rapid onset of drug action for respiratory as

well as non respiratory therapy.

iv. availability of a huge surface area for local drug action and systemic absorption of drugs.[2]

v. better tissue permeation of therapeutic molecules by pulmonary route in comparison with all the

other non injection routes of delivery (oral, buccal, transdermal and nasal), which were shown to

be not capable to allow the permeation of macromolecules unless penetration enhancers were

used. Furthermore, it must be pointed out that the enhancers, like surfactants and bile salts, may

cause significant irreversible tissue damages.[3]

Several formulations for pulmonary delivery are in various stages of development. Despite positive

results, conventional formulations have some limitations such as reduced bioavailability and side effects.

Nanocarrier systems in pulmonary drug delivery may be an alternative way to overcome the problems of

conventional formulations.

They allow the protection of therapeutic compounds from degradation, enhancing transepithelial

transport, reducing their immunogenicity, achieving relatively uniform distribution of drug dose among the

alveoli, improving solubility of the drug, controling drug pharmacokinetics and pharmacodynamics,

offering a sustained drug release which consequently reduces dosing frequency, improves patient

compliance, decreases incidence of side effects, and the potential of drug internalization by cells,

therefore increasing the bioavailability of drugs administered by pulmonary route for the treatment of

respiratory diseases (such as asthma, tuberculosis, chronic obstructive pulmonary disorder) and systemic

diseases (such as cancers, thrombosis, diabetes mellitus).

Nowadays, there has been an explosion in the number of nanocarriers which are defined as particles

having a size ranging from 1 nm to 1 µm [4]. To be suitable for pulmonary administration, these ones are

either made of lipids or composed of polymers. These systems are exploited for therapeutic purpose to

carry the drug in the body in a controlled manner from the site of administration to the site of action. This

allows the passage of the drug molecules across numerous physiological barriers.

4

Table 1:

The modern inhalation devices can be divided into three different categories: nebulizers, pressurized

metered dose inhalers (pMDI), and dry powder inhalers (DPI) [2]. In most cases, nanocarriers can be

delivered to the lungs by nebulization of colloidal dispersions or using pMDIs and DPIs in solid form.

These inhalers are based on different delivery mechanisms, and require different types of drug

formulations.

Localized therapy of the target organ generally requires smaller total doses to achieve clinically

effective results. In order to reach this goal, nanocarriers are engineered to achieve slow stimulation,

degradation and they have another advantage of being site specific targeted drug delivery.

Targeting mechanisms can be either passive or active. An example of passive targeting is the

preferential accumulation of chemotherapeutic agents in solid tumors as a result of the differences in the

vascularization of the tumor tissue compared with healthy tissue. For active targeting, the chemical

modification of the surface of drug carriers by using specific molecules enables them to be selectively

attached to diseased cells.

Two main aims of nanomedicine products currently in clinical trials are drug delivery devices and

diagnostic tests [5]. Nanocarrier systems have also been found useful to improve the performance of

imaging techniques applied for the in vivo diagnosis of tumors. Nanoscience and nanotechnology are thus

the basis of innovative delivery techniques that offer great potential benefits to patients and new markets

to pharmaceutical and drug delivery companies.

5

TYPES OF NANOCARRIERS FOR PULMONARY DRUG DELIVERY

LIPID MADE

Liposomes

Niosomes

Micro emulsions

Solid lipid nanoparticles

Lipidic micelles

POLYMER COMPOSED

Nanogels

Polymer micelles

Dendrimers

Polymer nanoparticles

Nanocapsules

Figure 1: Different types of nanocarrier systems.

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2. Respiratory tissue organization

The respiratory tract is like a tree. It is divided into two main parts: the upper respiratory tract

consisting of the nose, nasal cavity and the pharynx, and the lower respiratory tract consisting of the

larynx, trachea, bronchi and the lungs. Like the tree, the branches of the lung or the airways have thick

walls, and the alveoli or functional units of the lung have thin walls (0.1–0.5 µm) allowing possible access

of therapeutics to the systemic circulation. The surface area of the airways is approximately 0.25m 2,

smaller than the enormous alveolar surface area, which is approximately 100 m2, where the performance

of efficient gas exchange takes place [6]. Total lung capacity averages 6.7 l in men and 4.9 l in women.

Figure 2:

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3. Blood - air barriers

In order for a molecule to be absorbed from the lung into the blood it must pass through a number of

physiological barriers in the following order:

a. Surfactant: is the thin film of lipoprotein material covering the alveolar surface. It is a single

molecule thick monolayer of lung surfactant that spreads at the air/water interface.

b. Surface lining fluid: lies over the epithelium. This fluid acts as reservoir for lung surfactant and

appears to contain many of the components of plasma. In contrast to alveolar fluid, airway fluid contains

mucus which flows toward the trachea by the action of ciliated cells.

c. Epithelium: is the most significant barrier to absorption. This simple layer of cells varies from thick

columnar cells in the airways to extremely thin and broad cells in the alveoli.

d. Interstitium and basement membrane: The interstitium is the extracellular space inside tissues.

Epithelial and endothelial cells are attached to a tough but thin layer of interstitial fibrous material known

as the basement membrane.

e. Vascular endothelium: The final barrier to systemic absorption is another monolayer of cells that

make up the walls of small blood and lymph vessels. The permeability of this second cell barrier varies

with the type of blood vessel but even the tightest regions are thought to be more permeable to

macromolecules than the pulmonary epithelium.

Figure 3: The structure and components of

the blood- air barrier.

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3.1 Lung surfactant

Both airway and alveolar surface liquids are coated with at least a monolayer of highly surface active

lung surfactant in which the fatty acid tails of the surfactant lipids project into the air. The long chain

phospholipids that are the primary constituents of lung surfactant are water insoluble amphiphiles which

form liquid crystals but do not form micelles in aqueous media under physiological conditions. Lung

surfactant reduces the surface tension of lung surface liquids.

3.2 Epithelial surface fluids

Immediately below the molecular monolayer of lung surfactant lie the epithelial surface fluids through

which therapeutic molecules must diffuse to access to the epithelial cell layer. The relatively thick mucus

containing airway fluid that moves constantly towards the trachea with ciliary activi ty is distinct from the

thin alveolar fluid which contains no mucus and is not pushed by cilia.

Studies of human bronchoalveolar lavage samples suggest that the soluble proteins of surface fluids

resemble, with some important exceptions, the proteins found in serum in approximate serum proportions

and that some of the proportions change in various disease states [7]. Two immunoglobulins: IgG and IgA

are present in proportions that exceed levels seen in serum, but IgM, IgD and large nonimmunoglobulin

proteins are absent or present in very low amounts relative to serum levels. Transferrin was the only

nonimmunoglobulin protein that appears in higher proportion than in serum.

Figure 4: Under an electron microscope, the

barrier looks like this.

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3.3 Pulmonary epithelia

The next barrier to absorption after the surface fluid is the epithelium which in most cases is a single

monolayer of cells. The cells of the airway epithelium are very different from those of the alveolar

epithelium. There are over 60 cell types in the lung [8], the airway epithelium has at least four major cell

types, including the basal cell (the progenitor cell), the ciliated cell, the goblet cell and the Clara cell.

Figure 5: Types, structures and locations of different epithelial cells.

The alveolar epithelium is composed of only two major cell types, the extremely broad and thin Type I

cell and the small compact Type II cell (from which the Type I cell is thought to arise). Type I cells are

nonphagocytic, membranous pneumocytes. These cells are approximately 5 µm in thickness and

possess thin squamous cytoplasmic extensions that originate from a central nucleated portion. These

cells do not have any organelles and hence they are metabolically dependent on their central portion.

This reduces their ability to repair themselves if damaged.

10

Attached to the basement membrane are type II cells. These rounded, granular, epithelial

pneumocytes are approximately 10 to 15 µm thick. There are 6 to 7 cells per alveolus and these cells

possess great metabolic activity. They are believed to produce the surfactant material that lies the lung

and to be essential for alveolar repair after damage from viruses or chemical agents. The average human

alveolus has a surface area of 206 900 µm2 and is covered by 40 Type I cells and 67 Type II cells [8]. The

important roles of the lungs are: supplying oxygen, removing wastes and toxins, defending against hostile

intruders.

Figure 6: A cross section of an alveolar septum

showing a capillary and an interface between alveolar

type I cells and an endothelial cell.

3.4 The interstitium and basement membrane

The fourth potential barrier to absorption is the interstitium. The lung interstitium is the extracellular

and extravascular space between cells in the tissue. Within the interstitium are fibroblasts, tough

connective fibers which are collagen fibers, the basement membranes which serve as the structural

framework on which cells of the lung are mounted and interstitial fluid which slowly diffuses and

penetrates through the tissue.

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The epithelial and endothelial (capillary) cell layers in the lung are attached to a thin but tough matrix

of extracellular fibers called the basement membrane (a tough fibrous filter). The epithelial cells are

attached to one basement membrane and the capillary cells (endothelium) are attached to another.

Where these two cell layers come in contact their basement membranes fuse to form one common

basement membrane [9].

Figure 7: The structure of the interstitium and basement membrane.

3.5 Vascular endothelium

Molecules that are absorbed from the airspaces into the blood must traverse a final barrier after the

surfactant layer, surface fluid, epithelium and interstitium; the single cell monolayer that makes up the

walls of the blood vessels, the endothelium. In the lung, the huge surface area of the alveolar epithelium

is opposed by an equally large surface area afforded by a monolayer of capillary endothelial cells.

12

The surface area of a pulmonary endothelial cell is about 1/5 the size of a Type I cell. The basic

alveolar structure is the septum which is composed of capillaries sandwiched between two epithelial

monolayers and all held together by numerous extracellular and intracellular fibers (which are collagen

fibers, basement membranes, actin filaments and others) [10].

Normal leakage of plasma proteins from blood into the interstitium varies along the microvasculature

with the highest protein permeability thought to occur in the low pressure, post capil lary venules [11], in

general the vascular endothelium is thought to be quite leaky to proteins compared to the epithelium [12].

Figure 8: The morphology of the vascular endothelium as they lie beneath type I alveolar epithelial cells.

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4. Mechanism of deposition of particles in the lung

After administration, particles will undergo lung deposition. The deposition of inhaled particles in

different regions of the respiratory system depends on many factors, including the health of the patient,

the breathing rate, the respiratory volume, the humidity, and the geometry of the airways.

First of all, to reach the alveoli, the drug must be inhaled as particles with aerodynamic diameters

somewhere between 1 and 3 pm. The way the patient inhales, the type of formulation, the delivery device

used, the form and the size of the particles constituting the aerosol will have a considerable impact on the

eventual primarily deposition in the conducting airways or in the alveoli.

Depending on the particle size, airflow, and location in the respiratory system, particle deposition

occurs via one of the following principal mechanisms:

Impaction

Impaction occurs mostly in the case of larger particles that are very close to airway walls, near the first

airway bifurcations. Therefore, deposition by impaction is greatest in the bronchial region. Impaction

accounts for the majority of particle deposition on a mass basis.

Sedimentation

Sedimentation is the settling out of particles in the smaller airways of the bronchioles and the alveoli,

where the air flow is low and airway dimensions are small. Hygroscopic particles may grow in size as they

pass through the warm, humid air passages, thus increasing the probability of deposition by

sedimentation.

Interception

Interception occurs when a particle contacts an airway surface due to its physical size or shape.

Interception is most significant for fibers, which easily contact airway surfaces due to their length.

Furthermore, fibers have small aerodynamic diameters relative to their size, so they can often reach the

smallest airways.

Diffusion

Diffusion is the primary mechanism of deposition for particles less than 0.5 microns in diameter and is

governed by geometric rather than aerodynamic size. Diffusional deposition occurs mostly when the

particles have just entered the nasopharynx and in the smaller airways of the pulmonary system.

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Figure 9: The deposition percentages versus the particle sizes.

5. Mechanism of respiratory absorption

The extensive blood supply and the enormous surface area combined with an extremely thin barrier

between the pulmonary lumen and the capillaries, create conditions that are well suited for efficient

absorption. Soluble macromolecules can be absorbed from the lung into the body by two general

mechanisms. They can either pass through the cells (absorptive transcytosis) or between the cells

(paracellular transport) [13].

Absorptive transcytosis may occur independent of a plasma membrane receptor (transcellular

transport) or it may involve receptor mediated binding followed by vesicular transport (receptor mediated

transcytosis). Paracellular transport is usually thought to occur through the junctional complex between

two cells. A second type of paracellular transport may occur at the junction of three cells which presents

at specific spots on the circumference of both endothelial and epithelial cells. A third type of paracellular

transport may occur when a cell dies and sloughs leaving a relatively large pore on the basement

membrane.

Distributed on the surface of different cell types in the lung are peptidases which play an essential role

in cell and tissue growth, differentiation, repair, remodeling, cell migration and peptide mediated

inflammation in the respiratory tract [14]. Peptides that have been chemically altered to inhibit peptidase

enzymes exhibit very high absorptive capability by the pulmonary route. The ends of their amino acid

chains are often tucked into the globular structure of the protein and are not available for hydrolysis. The

large proteins may preclude their fit into catalytic clefts of the enzyme structure. The use of blocking

chemistry can combine ineffective peptides with enzyme inhibitors and create great medical value [15].

15

As previously mentioned, it appears that the alveolar epithelium is considered as the main barrier to

absorption [16]. This is composed of polarized cells, permeable to water, gases and lipophilic molecules.

However, the permeation of hydrophilic substances of large molecules and ionic species is limited [15]. It is

known that the absorption rate of various proteins across the epithelium is size dependent, particularly in

case of paracellular mechanism.

The bioavailability of peptides and proteins is 10-200 times greater by pulmonary route as compared

with other routes. Inhalation is a fast way for proteins to get into the body because drug efflux transporters

and metabolizing enzymes are present in the lung at much lower levels than in the gastrointestinal tract

and the liver. Lipophilic small molecules are absorbed extremely fast with t1/2 absorption being

approximately 1-2 minutes. Hydrophilic small molecules are absorbed rapidly with t1/2 absorption being

approximately 65 minutes.

Insoluble molecules that slowly dissolve after inhalation may stick in the lung for many hours or even

days. In this case, encapsulation of such slow absorptive molecules by using nanoparticles and

liposomes can help to control absorption.

Figure 10: Transport processes from the alveolar lumen to the capillary side.

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5.1 Transport through cells: Transcytosis

Transcytosis is the mechanism that cells use to transport molecules from one side of the cell to the

other without disrupting the barrier function of the plasma membrane or its electrochemical potential on

either side of the cell. This kind of transcytosis is independent of plasma membrane receptors

(transcellular) [17].

In the permeation of proteins of higher size, the receptor mediated endocytosis appears to be more

involved. Peptides and peptidomimetics drugs can be absorbed by active transport using the high affinity

peptide transporter (PEPT2) existing in alveolar type II cells and in capillary endothelium. The presence of

caveolin in alveolar type I cells and endothelium of the lung and clathrin coated vesicles in alveolar type I

and type II cells suggests the possible involvement of such pathways in the absorption of large size

proteins.

5.2 Paracellular transport: Diffusion between cells

Peptides and proteins of low molecular weight (≤ 40 kDa) cross the alveolar epithelium mainly by the

paracellular pathway. This also prevails when there is epithelial injury such as edema or inflammation.

This mechanism occurs through the junctional complex between cells.

a. Tight Junctions: complex structures of multiple proteins which serve as intricate and dynamic

fasteners of cells to each other. There are approximately 60 miles of cell junction in human airways and

over 2000 miles in the alveolar region. The tightness or leakiness of a cell junction is thought to correlate

with the number and continuity of rows or strands in the junctional web [18].

b. Endothelial junctions: the pulmonary endothelial cell barrier is relatively permeable to

macromolecules compared to the epithelium and probably does not limit protein absorption from the

airspaces. The most tenuous junctions are seen in the venular endothelial cells which are known to line

the walls of the leakiest portion of the vascular system.

c. Epithelial junctions: The structures of pulmonary epithelial junctions are markedly different from that

of endothelium. They are more elaborate with more rows, and the rows are continuous. The mesh is more

open on the bottom of the junctions between Type I and Type II cells as compared to the junctions

between two Type I cells [19].

d. Permeability increases in smokers and in most of pulmonary disease states: Cigarette smoke

contains thousands of different compounds, some of which are assumed to stimulate alveolar

macrophages and polymorphonuclear cells to release oxidants which are thought to damage the

epithelium (damage to alveolar Type I cells adjacent to the bronchioalveolar junctions). Smoker’s lungs

are much more permeable to small solutes and proteins like albumin and insulin than non-smoker’s.

Early studies suggested that cigarette smoke increased permeability by opening tight junctions [20].

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e. Osmolality affects absorption of solutes: Soluble dry powder aerosols, upon landing in the surface

lining fluid of the alveoli, probably stimulate the rapid secretion of water to equilibrate any hypertonic

solution that results from dissolution of the powder in the small volume of surface lining fluid. Pure water

placed into the lung is very rapidly absorbed [21] and with it, by bulk flow, large and small solutes including

peptides are also carried rapidly into the body. Pure water probably also opens tight functions widely,

thereby enabling absorption of bulk fluid with solutes dissolved therein.

6. Significance of nanocarrier systems for pulmonary administration

The successful integration of novel drugs with devices capable of delivering defined doses to the

respiratory tract in a variety of ways: via aerosols, metered dose inhaler (MDI) systems, dry powder

inhalers (DPI) and solutions (nebulizers).

Nanocarriers such as micelles, liposomes, nanoparticles, and microemulsions can incorporate a

variety of therapeutics and present several advantages for drug delivery to the lung, including controlled

release, protection from metabolism and degradation, decreased drug toxicity, enhanced cellular binding

and uptake, targeting capabilities, and reduction of side effects. All of these properties contribute to

increase bioavailability of pharmaceutical products, and therefore increase the efficiency of the treatment.

Figure 11: Various types of nanocarrier systems with their relative sizes.

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6.1 Micelles as nanocarriers in pulmonary delivery

Colloidal systems, such as micellar solutions, vesicle and liquid crystal dispersions, as well as

nanoparticle dispersions consisting of small particles of 10-500 nm in diameter show great promise as

carriers in pulmonary drug delivery systems. They demonstrate optimal drug loading and release

properties, long shelf life and low toxicity.

Micelles are nanostructures resulted from the self assembly of amphiphilic macromolecules in

aqueous environment. Drugs can be trapped in the core of a micelle and transported at concentrations

even greater than their intrinsic water solubility. A hydrophilic shell can form around the micelle, effectively

protecting the contents. In addition, the outer chemistry of the shell may prevent recognition by the

reticuloendothelial system, and therefore prevent early elimination from the bloodstream. A further feature

that makes micelles attractive is that their size and shape can be changed. Chemical techniques using

cross linking molecules can improve the stability of the micelles and their temporal control. Micelles may

also be chemically altered to selectively target a broad range of disease sites.

Polymeric micelles can encapsulate water insoluble drugs, proteins and DNA, and target the

therapeutics to their site of action in an active or passive way. Polymeric micelles share many structural

and functional features with natural transport system, such as viruses and lipoproteins.

Figure 12: The incorporation of drug molecules into the micelles.

19

The effect of chemical manipulations on the encapsulation, release, biodistribution and cellular

interaction of the polymeric micelles is assessed to select appropriate methods for optimized delivery of

P-glycoprotein substrates to resistant tumors. Another focus of this nanometric dosage form is to improve

the solubilization of a poorly water soluble drug intended to be administered via pulmonary route.

The advantages of polymeric micelles are better stability than surfactant micelles, the ability to

solubilize an adequate amount of hydrophobic drugs, prolonged circulation times in vivo, and the

capability to accumulate in the target organs. In addition, drug loaded polymeric micelles are strongly

suggested to pass through the mucus layer associated with bronchial inflammatory diseases directly to

their receptors in the epithelial cells.

6.2 Liposomal pulmonary drug delivery vehicles

Liposomes are one of the most extensively investigated systems for controlled delivery of drug to the

lung [22]. Liposomes are small spherical vesicles composed of one or more bilayers of phospholipids,

cholesterol, lung surfactants and synthetic lipids.

Owing to their structure, they allow the incorporation of hydrophilic drugs in the aqueous core, and

hydrophobic drugs within the lipid bilayer. Depending on the number and composition of the bilayers and

the coating present, it is possible to obtain systems with modified release characteristics.

Figure 13: Liposomal formulations can encapsulate many kinds of drugs.

20

The utilization of liposomal drug formulations for aerosol delivery to the lung has many potential

advantages, including aqueous compatibility, sustained pulmonary release to maintain therapeutic drug

levels and facilitated intracellular delivery particularly to alveolar macrophages.

Owing to their interaction with endogenous phospholipids, liposomal formulations also promote an

increased retention time in the lungs. Furthermore, the use of phospholipids similar to the surfactant

promotes the absorption of the incorporated drugs.

In addition, liposomal formulations may prevent local irritation and reduce toxicity both locally and

systematically. These results suggest that liposomal aerosols should be more effective for delivery,

deposition, absorption and retention of water insoluble compounds in contrast to water soluble

compounds.

The development of liposomal formulations for aerosol delivery with jet nebulizers has expanded the

possibilities for effective utilization of aerosol based therapies in the treatment of several respiratory and

non respiratory diseases.

Liposomal formulations have been proposed to delivery anticancer drugs, corticosteroids,

immunosuppressants, antimycotic drugs, antibiotics for local pulmonary infections and cystic fibrosis and

opioid analgesics for pain management. Many of them have reached the stage of clinical trials for the

treatment of several pulmonary diseases [23]. High drug encapsulation efficiency was obtained and it gives

an advantage to solve the problem of multi drug resistance in case of tuberculosis [24]. A new trend in

vesicles lung delivery is addressed to obtain efficient and safe vaccine delivery systems [25].

Moreover, the use of liposomes for pulmonary administration of several drugs including peptides,

therapeutic proteins and DNA for gene therapy has been suggested. Gene therapy is currently being

developed for a wide range of acute and chronic pulmonary diseases, including cystic fibrosis, cancer and

asthma.

A highly effective nanocomposite aerosol consisting of a biodegradable polymer core, and an efficient

and safe cationic lipid, was proposed to prepare delivery systems called cationic liposomes for pulmonary

gene delivery [26].

Much interest has focused on cationic liposomes for pulmonary gene therapy because cationic

liposomes offer the advantage of self assembly with DNA material through favorable cationic–anionic

electrostatic interactions. Additional advantages include the evasion from complement inactivation after in

vivo administration, the low cost, the relative ease in producing nucleic acid liposome complexes in large

scale, and the exhibition of low cytotoxicity [27].

21

Figure 14: Liposomal formulations are also applied for gene therapy.

6.3 Microemulsions in pulmonary administration

These dosage forms show numerous advantages as compared to other drug targeting systems, such

as easy manufacture and maximum drug incorporation. Due to their physicochemical characteristics,

reverse emulsions and microemulsions allow solubilizing a large amount of hydrophilic drugs. Reverse

miroemulsions are stabilized by lecithin and they use propane or dimethylether as propellants.

These microemulsions, characterized by mean geometric diameters ranged between 1 and 5 µm and

by a respiratory fraction up to 36%, showed high stability during more than 4 weeks at room temperature.

Water-in-HFA (hydrofluoroalkane) emulsions stabilized by non-ionic fluorinated surfactants have been

also studied in order to administer drugs by pulmonary route.

Emulsion systems have been introduced as alternative gene transfer vectors to liposomes. Many

emulsion studies for gene delivery have shown that the binding of the emulsion/DNA complexes was

stronger than in case of liposomal carriers [28].

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7. Micro- and nanoparticle delivery systems

7.1 Microspheres as pulmonary drug carriers

These biodegradable formulations consisting of an oily core surrounded by a thin polymeric matrix

(derived from natural or synthetic polymers) have been largely used as drug targeting systems via

different routes. Both hydrophilic and lipophilic therapeutic molecules can be capsulated or incorporated

into microspheres.

Compared to liposomes, microspheres have an in vivo and in vitro more stable physicochemical

behaviour and allow a slower release and a longer pharmacological activity of the encapsulated drugs.

Biodegradable microspheres are prepared by using varied polymers, including albumin, chiotosan,

polysaccharide, or polylactic acid.

Pulmonary administration of aerosolized microspheres allows a sustained and prolonged release of

drugs, because the drugs are being protected against the enzymatic hydrolysis. Microspheres are less

hygroscopic and hence less liable to swell in the presence of moisture located in the lung. Different drugs

have been encapsulated into microspheres including corticosteroids, viruses, proteins, tuberculostatics

and anticancer agents.

7.2 Polymeric nanoparticles

Nanoparticles present the same characteristics as the microspheres: they are also constituted of a

polymeric matrix and drugs bound either at the surface of the particles or encapsulated into the vectors.

They can be constructed by polymerization of monomers or by polymer dispersion.

Figure 15:

Polymeric

nanoparticles.

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Among synthetic polymers, poly (lactic-co-glycolic acid) (PLGA) is well studied and widely used in

biomedical applications owing to its biocompatibility, biodegradation, protection against the enzymatic

degradation, surface modification capability, low cost, non toxicity and usefulness in production of

modified release formulations.

Large porous PLGA particles showed to be more efficient for the pulmonary drug delivery of inhaled

particles than small porous or nonporous particles did. The fact is large porous particles aggregate less

and deaggregate more easily under shear forces than small and nonporous particles do, hence they

appear to more efficiently aerosolized from a given inhaler device than conventional therapeutic particles.

These targeting systems can be designed for in vivo applications including molecules with therapeutic

activities and radiocontrast agents or in vitro as a support for molecules intended for diagnosis.

Figure 16: Nanoparticles can be designed for drug delivery systems as well as disease diagnosis.

7.3 Solid lipid nanoparticles

These dosage forms consist of a solid lipid matrix at both ambient and body temperatures, dispersed

in an aqueous solution and stabilized with a layer of emulsifying agents (usually phospholipids).

They combine the advantages of the biocompatibility of liposomes and the possibility of industrial

scale up of polymeric nanoparticles. Indeed, they emerged as alternatives to liposomes and

pharmaceutical emulsions, because they are more stable in biological fluids and during storage. They are

also less toxic, and hence higher tolerability than polymeric nanoparticles owing to their biocompatibility

and biodegradability.

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The key parameters determining the encapsulation rate of drugs into lipids are the solubility or the

miscibility of drugs into melted lipids, the chemical and physical structures of the lipid solid matrix, and the

polymorphous state of the lipids.

Three possible loadings of drugs can be envisaged:

The dispersion of drugs into the solid lipid particles

The core-membrane model containing a membrane conjugated with the drug

The core-membrane model containing a core conjugated with the drug.

Figure 17: Solid lipid nanoparticles.

7.4 Dendrimer based nanoparticles

These carriers are polymers, which have hyperbranched structures, with layered architectures. The

research in dendrimer mediated drug delivery has mainly been focused on the delivery of DNA drugs into

the cell nucleus for gene or antisense therapy. Many studies have been reported on the possible use of

dendrimers as non viral gene transfer agents [29].

In addition, it is suggested that dendrimers could be viable carriers for pulmonary delivery of low

molecular weight heparin (LMWH) through electrostatic interactions in preventing deep vein thrombosis [30]. Several studies have been published regarding pulmonary applications of dendrimers as systemic

delivery carriers for macromolecules.

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8. Application of nanocarrier systems in treatment of non respiratory diseases

In 1993, the US Food and Drug Administration (FDA) approved the first protein administered via

inhalation is the recombinant human enzyme called desoxiribonuclease, also known as Dornase α

(Pulmozyme®, Genentech Inc., CA, USA), for the treatment of cystic fibrosis [31]. Currently, there are

several peptides and proteins with therapeutic potentials such as insulin, calcitonin, cyclosporin A or

interferon-γ, for which pulmonary administration is under development and in clinical trials.

Treatment of diabetes mellitus

Of all the proteins under development, insulin has been the target of most studies. There are many

formulations have been developed for administering insulin by pulmonary route. Such formulations are in

various stages of development, with one approval for marketing by the FDA and European Medicines

Agency (EMA) is the Exubera (Exubera®, Pfizer, NY, USA).

In general, patients who received inhaled insulin formulations demonstrate better therapeutic effects,

such as faster onset of action, lower weight gain, lower incidence and severity of hypoglycemia and

greater satisfaction (higher comfort and convenience) as compared to patients receiving subcutaneous

injection of regular insulin.

Figure 18: The Exubera is now no longer in the

market for treatment of diabetes mellitus

because of many side effects.

EXUBERA® INHALER

Although the results from clinical trials demonstrated the usefulness of inhaled insulin to control blood

glucose level, the conventional formulation Exubera have been withdrawn because of many side effects

26

such as increased anti-insulin antibodies, and the possibility to suffer from irreversible pulmonary

damage.

Another inhaled insulin product, the Afrezza™, is in the late stage of clinical trials, and currently under

review by the FDA for the treatment of type l and type II diabetes [32]. The Afrezza™ is a novel, and

extremely rapid acting insulin comprising Technosphere® insulin powder in unit dose cartridges for

administration with the inhaler. The Afrezza™ appears to overcome several limitations of the Exubera®.

Treatment of bone metabolic disorders

Calcitonin is used in the clinical treatment of musculoskeletal disorders such as osteoporosis, Paget's

disease, hypercalcemia and bone metastasis [33]. Pulmonary administration presents higher bioavailability

of peptide and protein therapeutics. Salmon calcitonin inhalation has been found effective in the treatment

of osteoarthritis in a limited number of clinical studies [34]. The inhaled formulation did not cause serious

damage or local irritation to the pulmonary epithelium.

Treatment of immune diseases

Cyclosporin-A is used for treatment of immune diseases such as pulmonary chronic asthma,

hypersensitivity, bronchiolitis, sarcoidosis or the treatment of lung transplant rejection [35]. Pulmonary

delivery of cyclosporin-A seems to be a viable alternative to parenteral delivery. The reason is, as a local

administration, it will reduce the long term nephrotoxicity of oral and parenteral administration.

Furthermore, it allows a higher concentration of cyclosporin-A at the site of action. Cyclosporin-A has also

been created and evaluated in the laboratory and in early clinical studies.

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Figure 19: Clinical applications of nanoparticles for drug delivery to the respiratory tract are still in early

stages of development.

Treatment of respiratory diseases

Interferon-γ plays a key role in establishing and maintaining protective immune responses and host

defense against a variety of microorganisms, including mycobacteria via macrophage activation,

especially in patients suffering from human immunodeficiency virus (HIV) [36]. Interferon-γ is still in phase II

of clinical trials, testing the safety and effectiveness of the inhaled interferon-γ for the treatment of a lung

infection caused by a bacterium called Mycobacterium avium complex.

Treatment of renal cell carcinoma and advanced melanoma

Interleukin-2 is used in renal cell carcinoma, melanoma and diseases characterized by states of

immunodeficiency. Intravenous administration of interleukin-2 is associated with severe and dose limiting

side effects in the kidneys, the cardiovascular system and the liver. It has been studied for respiratory

administration and it is currently the subject of clinical trials, obtaining positive results, especially in

reducing associated side effects and increasing the median survival of patients.

Treatment of cancers by chemotherapy

There is a large amount of published data regarding aerosol delivery of chemotherapy in cancer cell

cultures, animal models, and Phase I/II human studies. The first chemotherapeutic agent, investigated

almost 30 years ago, was 5-fluorouracil (5-FU) [37]. Although some phase I/II studies are already being

carried out, clinical trials to determine the effects of such formulations in delivery of anticancer drugs in

humans are still awaited.

Table 2: Public studies with inhaled chemotherapy regimens are currently in clinical trials [38].

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DOX: doxorubicin; CIS: cisplatin; PTX: paclitaxel; 9NC: 9-nitro-camptothecin;

CARBO: carboplatin; 5-FU: 5-fluorouracil.

Treatment of infections by antibiotics

Aerosolized tobramycin (Tobi®) was approved by FDA in 1997 as the first nebulized antibiotic.

Aerosolized aztreonam lysine (Cayston®, Gilead Seattle, WA) was approved in 2010 for patients with

cystic fibrosis and chronic pseudomonas infection [39].

Colistin (Coly-Mycin®) is a polypeptide antibiotic of the polymycin class. This inhaled antipseudomonal

antibiotic is not yet FDA approved for nebulization. Inhaled amikacin (Arikace®, Transave) is a liposomal

formulation of amikacin that is different from the common intravenous formulation.

Phase II trials began on inhaled ciprofloxacin in 2007. Nebulized liposomal ciprofloxacin is being

developed for both cystic fibrosis and bronchiectasis. MP-376 (Aeroquin) is a new formulation of

levofloxacin, which is being developed by Mpex Pharmaceuticals for aerosol administration for the

management of chronic pulmonary infections due to Pseudomonas aeruginosa and other bacteria.

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Table 3: Liposomal delivery of antibiotics [40].

9. Pulmonary Toxicity of Nanocarriers

Despite the many advantages presented by the nanocarriers, they still remain some limitations such

as induction of inflammatory responses and epithelial damage in the lungs, as well as extrapulmonary

effects, including oxidative stress or increased blood clotting [41].

Upon the interaction with the nanoparticles, functions of the thin film lung surfactant may be

compromised by these inhalable nanoparticles, and that may cause life threatening consequences.

In the case of lipid carriers, they are composed of physiological components, and the body has

metabolic pathways that can reduce the toxic effects derived from short and long term exposure to the

lipid carriers. For these reasons, vesicular nanocarriers, consisting of lung surfactants and/or synthetic

amphiphiles may provide an efficient delivery system due to their biocompatibility, biodegradability and

30

non-toxic nature [42]. However, it is necessary to take into account the emulsifiers and preservatives used

in these formulations.

Thomas et al. [43] investigated the effects of soy phosphatidylcholine liposomes on pulmonary function

in healthy adults. The results showed that there are no adverse effects on lung function, as well as

oxygen saturation, and the liposomes used in the study were well tolerated.

The adverse health effects seem to be dominated by pulmonary symptoms. For instance, many

reports have addressed that occupational exposure of inhaled rigid nanoparticles can lead to respiratory

diseases such as pneumoconiosis (pulmonary fibrosis) and bronchitis, and increase the risk of chronic

obstructive pulmonary disorder.

Despite the evolution that has occurred in recent years, only a few products based on nanotechnology

are now on the pharmaceutical market. However, it is expected in the near future that an increase in the

development of formulations containing nanocarriers will improve the properties of various drugs, such as

higher bioavailability, controlled release and targeting to specific organs and tissues.

This approach also allows the administration of stable drugs, as well as insoluble hydrophobic drugs

that otherwise would be excluded during the pharmaceutical development despite their high therapeutic

potential.

It is also expected that the development of new and improved delivery devices will allow us to

overcome the limitations of pulmonary administration of drugs, with regards to the non reproducibility of

the dose.

10. Conclusion

One of the most important routes for local and systemic delivery of drugs is the pulmonary route.

Owing to the physiology of the lungs, inhalation is presented as a promising noninvasive alternative to

parenteral administration of several drugs.

Techniques and new drug delivery devices intended to deliver drugs to the lungs have been widely

developed. Nowadays, the direct application of a drug by inhalation therapy uses pressurized metered

dose inhalers (pMDI), dry powder inhalers (DPI) and nebulizers.

To convey a sufficient dose of drug to the lungs, suitable drug carriers are required. The application of

nanotechnology in medicine has been the target of growing interest over recent years. Nanocarriers,

including nanoparticles, liposomes and micelles, may be used for the incorporation of drugs allowing their

31

protection from degradation, targeting to desired organs or tissues and the reduction of side effects. In the

case of protein formulations, the use of nanocarriers have several advantages such as improved stability

and transepithelial transport, obtaining modified release formulations, deep penetration in tissues,

increased internalization by cells, high strength, and their ability to escape the in vivo defensive system

and thus increase systemic circulation time. They also allow the reduction of the immunogenicity of the

proteins, thus decreasing the toxicity of the formulations.

In addition, these systems make it possible to use relatively small numbers of vector molecules to

deliver substantial amounts of a drug to the target. Nanoparticle delivery to the lungs is an attractive

concept because it can cause retention of the particles in the lungs accompanied with a prolonged drug

release, drug protection and improved bioavailability over the conventional pulmonary drug delivery

systems.

Aerosol administration of the therapeutics to the pulmonary epithelium for systemic delivery represents

a significant opportunity for many classes of drugs and applications, including anti-tumor therapy, gene

therapy, AIDS therapy, radiotherapy, in the delivery of macromolecules as peptides and proteins or small

molecules as antibiotics, virostatics and vaccines. Advantages of aerosol administration include: more

rapid absorption into the systemic circulation, and higher bioavailability.

Currently, the main application areas of nanomedicine are imaging and cancer therapy, however,

studies have been carried out in various areas, such as peptide and protein delivery, vaccination, gene

therapy, tissue engineering or production of devices for administration of drugs.

There are still many challenges that are being faced. Further research efforts are needed to ensure

the safety of long-term in vivo applications and the development of scale up from laboratory to industry in

order to reach, within a few years, the safety and large-scale production at affordable costs of innovative

pulmonary delivery medicines.

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ACKNOWLEDGEMENT

This dissertation would not have been possible to be done without the guidance and the help of two

individuals, who contributed and extended their valuable assistance in the preparation as well as the

completion of this diploma work.

First of all, I am deeply grateful to my supervisor, Dr. Miklós Vecsernyés, PhD, Dean of Faculty of

Pharmacy, University of Debrecen. His encouragement, guidance and support from the initial to the final

level enabled me to develop an understandable and completed research work.

Another person who infused spirit to me, gave me strength and courage to overcome the obstacles, is my

beloved man, Mr. Liem Nguyen. He helped me a lot in checking, fixing and correction of the thesis.

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In all my sincerity, I will never forget their great work. Thank you!

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