Introductory Aspects of Soft Nanoparticles

JOAN ESTELRICH, MANUEL QUESADA-PÉREZ, JACQUELINE FORCADA AND JOSÉ CALLEJAS-FERNÁNDEZ

1.1 Nanoparticles

Nanotechnology is the science that deals with matter at the scale of 1 billionth of a metre (i.e. 10-9 m = 1 nm) and is also the study of manipulating matter at the atomic and molecular scale. A nanoparticle is the most fundamental component in the fabrication of a nanostructure and is far smaller than the world of everyday objects that are described by Newton's laws of motion, but larger than an atom or a simple molecule that are governed by quantum mechanics.

According to the definition of the International Organization for Standardization (ISO), a nanoparticle is a particle whose size spans the range between 1 and 100 nm. Metallic nanoparticles have different physical and chemical properties from bulk metals (e.g. lower melting points, higher specific surface areas, specific optical properties, mechanical strengths and magnetizations), properties that might prove attractive in various industrial applications. However, how a nanoparticle is viewed and is defined depends very much on the specific application. In this regard, for biomedical applications, structures and objects up to 1000 nm in size are included as nanostructured materials used in medicine.

Of particular importance, optical properties are among the fundamental attractions and characteristics of a nanoparticle. For example, a 20 nm gold nanoparticle has a characteristic wine-red colour, a silver nanoparticle is yellowish grey and platinum and palladium nanoparticles are black. Not surprisingly, the optical characteristics of nanoparticles have been used for centuries in sculptures and paintings even before the fourth century AD. The most famous example is the Lycurgus cup (fourth century AD). This cup, at present in the British Museum in London, is the only complete historical example of a special type of glass, known as dichroic glass, that changes colour when held up to the light. When it is looked at in reflected light or daylight, it appears green. However, when light is shone into the cup and transmitted through the glass, it changes colour to red. This property puzzled scientists for decades and the mystery was not solved until 1990, when researchers in England scrutinized broken fragments under a microscope and discovered that Roman artisans were nanotechnology pioneers: they had impregnated the glass with a very small quantity of minute (~70 nm) colloidal silver and gold in an approximate molar ratio of 14:1, which gives it these unusual optical properties.

Gold suspensions were familiar to alchemists in the Middle Ages and the reputation of soluble gold was based mostly on its fabulous curative powers against various diseases, for example, heart and venereal diseases, dysentery, epilepsy and tumours. Metallic nanoparticles were used in mediaeval stained glasses. The mediaeval artisans trapped gold nanoparticles in the glass matrix in order to generate ruby-red colour in windows. They also trapped silver nanoparticles, which gave the glass a deep-yellow colour. Beautiful examples of these applications can be found in glass windows of many Gothic European cathedrals.

In the seventeenth century, the so-called Purple of Cassius was highly popular. It was a colloid made by reducing a soluble gold salt with stannous chloride. It was used as a colorant and to determine the presence of gold as a chemical test. The first scientific study of gold particles was carried out by Faraday in 1857. He observed that gold suspensions with a ruby-coloured appearance, made by reducing an aqueous solution of chloroaurate (AuCl4-) with phosphorus in CS2 (a two-phase system), changed their colour from red to blue upon heating or addition of salt. Faraday correctly attributed the colour change to an increase in the effective particle size caused by aggregation. Since that pioneering work, thousands of scientific papers have been published on the synthesis, modification, properties and assembly of metal nanoparticles, using a wide variety of solvents and other substrates. Nowadays, the most widely used nanotechnology product in the field of in vitro diagnostics is colloidal gold in lateral flow assays, which is used in rapid tests for pregnancy, ovulation, human immunodeficiency virus (HIV) and other indications. Gold nanoparticles were introduced into these tests in the late 1980s because gold conjugates have particularly high stability, which is critical for avoiding false positives.

In 1959, Richard Feynman gave a talk entitled 'There's plenty of room at the bottom', where he predicted the new things and new opportunities that one could expect in the very small world. Norio Taniguchi of Tokyo University of Science was the first to propose in 1974 the term 'nanotechnology'. The age of nanotechnology had begun.

The activity in the field of the nanotechnology has grown exponentially worldwide during the past three decades, becoming a major interdisciplinary area of research. This growth has been driven to a great extent by the integration of nanotechnology into the field of medical science, since nanostructured materials have unique medical effects. The control of materials in the nanometric range not only results in new medical effects but also requires novel, scientifically demanding chemistry and manufacturing techniques. This definition does not include traditional small-molecule drugs as they are not specifically engineered on the nanoscale to achieve therapeutic effects that relate to their nanosize dimensions. Nanoparticles have numerous functional moieties on their surfaces capable of multivalent conjugation for diagnostic, targeting, imaging and delivery of therapeutic agents (Figure 1.1).

Owing to their unique characteristics, including large surface area, structural properties and long circulation time in blood compared with small molecules, nanoparticles have emerged as attractive candidates for optimized therapy through personalized medicine. Potential advantages of engineered therapeutic nanoparticles are the ability to convert unfavourable physicochemical properties of bioactive molecules to desirable biopharmacological profiles, to improve the delivery of therapeutic agents across biological barriers and compartments, to control the release of bioactive agents, to enhance therapeutic efficacy by selective delivery of drugs to biological targets and to perform theranostic functions by combining multimodal imaging and simultaneous diagnosis and therapy into multifunctional nanoplatforms.

A handful of nanomaterials and nanoparticles are being studied in clinical trials or have already been approved by the US Food and Drug Administration (FDA) for use in humans and many proof-of-concept studies of nanoparticles in cell-culture and small-animal models for medical applications are under way. Examples of such nanoparticles and nanomaterials are provided in Table 1.1.

As can be deduced from Table 1.1, there is a plethora of nanoparticles suitable for biomedical applications. This large number of nanoparticles is due to recent developments in synthetic methods, which mostly involve polymeric formulations, inorganic formulations or a combination of both. The resulting organic–inorganic hybrid materials inherit properties both of the polymers and of the metallic compounds.

Magnetoliposomes (liposomes encapsulating iron nanoparticles) are the most relevant example of hybrid nanoparticles. In this book, however, we will restrict ourselves to soft nanoparticles. More specifically, our choice is a compromise between well-known particles (e.g. polymeric micelles) and new particles whose potential is exciting but not yet widely proved.

1.2 Soft Nanoparticles

Classical micelles were the type of soft nanosystems used in pharmaceutical applications long before the emergence of nanotechnology. As a result of micelle formation, an organic compound that would normally be insoluble in water can be 'dissolved' in a surfactant solution because it can move into the oily interior of the micelle. This phenomenon, known as solubilization, has been used for solubilize drugs. As a few representative examples, phenolic compounds are frequently solubilized with soap to form clear solutions, which are widely used for disinfection. Non-ionic surfactants are efficient solubilizers of iodine. Such iodine–surfactant systems (referred to as iodophors) are more stable than iodine–iodide systems. On the other hand, the low solubility of steroids in water presents a problem in their formulation for ophthalmic use. The requirement for optical clarity precludes the use of oily solutions or suspensions. The use of non-ionic surfactants permits the production of clear solutions, which are stable to sterilization. This type of surfactant has also been used to solubilize essential oils and water-insoluble vitamins.

Among the first nanotechnology drug-delivery systems were lipid vesicles, which were described in the mid-1960s and later became known as liposomes. At first, they were used to study biological membranes; several practical applications, most notably in drug delivery, emerged in the 1970s (in this period, Donald Tomalia invented, named and patented the dendrimers, although they were not used as drugs until 1980s). Soon, however, it was observed that liposomes suffered an important drawback when used as carriers for therapeutically active compounds: they undergo rapid degradation due to the macrophage phagocyte system (MPS). As with all foreign colloidal particles, liposomes are quickly recognized as 'non-self' and taken up by the cells of the MPS, chiefly macrophages in the liver and spleen. This leads to the inability to achieve sustained drug delivery over a prolonged period of time. The incorporation in the bilayer of cholesterol and, mainly, biocompatible, hydrophilic polymers with a flexible main chain, such as poly(ethylene glycol) (PEG), led to long-circulating liposomes, also known as sterically stabilized liposomes or Stealth liposomes. Doxil, the first nanomedicine to secure regulatory approval by the FDA (for the treatment of AIDS-associated Kaposi's sarcoma in 1995 and in Europe in 1997 with the brand name Caelyx), was obtained by encapsulating doxorubicin within liposomes. At present, liposomes are the most commonly used soft nanoparticles for clinical applications, especially in the treatment of cancer and systemic fungal infections. The number of liposomal products on the market or in advanced clinical studies exceeds two dozen. Apart from liposomes, polymer-based nanoformulations constitute the majority of the nanoparticle therapeutic agents available for clinical use. Polymer–drug conjugates are another extensively studied nanoparticle drug-delivery platform currently in clinical practice. Many polymers have been proposed as drug-delivery carriers, but only a few of them with linear architecture have been accepted into clinical practice. PEG was first introduced into clinical use in the early 1990s. Today, there are around a dozen examples of PEGylated drugs in clinical practice. Other macromolecule–drug conjugates have also been developed as drug carriers, such as Abraxane, a 130 nm albumin-bound paclitaxel that was approved by the FDA in 2005 as a second-line treatment for patients with breast cancer.

Biodegradable polymeric micelles with a size of 10–200 nm have attracted considerable attention as drug-delivery nanocarriers and have shown remarkable therapeutic potential. Polymeric micelles are formed by self-assembly of block copolymers consisting of two or more polymeric chains with different hydrophobicity. These copolymers spontaneously assemble into a core–shell micellar structure in an aqueous environment to minimize the Gibbs energy.

When short-chain phospholipids were combined with long-chain phospholipids, structures closely related to liposomes and also with micelles were found: the bicelles. They were used in the 1990s as a model membranes well suited to magnetic resonance studies of membrane protein structure because of their ability to orient in a magnetic field, and have been used in most NMR structural studies of transmembrane proteins. As a biomedical tool, the use of bicelles has been proposed to favour the penetration of encapsulated drugs through the corneum stratus of the skin.

Dendrimers have emerged as another novel class of drug-delivery soft nanoparticle platform because of their well-defined architecture and unique characteristics. The specific molecular structure of dendrimers enables them to carry various drugs using their multivalent surfaces through covalent conjugation or electrostatic adsorption. Alternatively, dendrimers can be loaded with drugs using the cavities in their cores through hydrophobic interaction, hydrogen bonds or chemical linkages.

As mentioned previously, there are promising candidates for nanoparticles whose clinical applications are still limited. Among them are polymer-based soft nanoparticles, which are very interesting for use as nanosized drug carriers. An ideal drug carrier needs to combine both the targeting property and the stimulus responsiveness to enhance the bio-availability of the drug together with the reduction of side effects. Therefore, the design of stimuli-responsive nanoparticles for drug delivery to release the drug in a controlled way when arriving at the targeted site is highly desirable. Among polymer-based soft nanoparticles there are stimulus-responsive nanoparticles or environment-sensitive nanoparticles having the ability to change their size or volume when exposed to external changes or signals. These nanoparticles are also known as micro/nanogels. Stimuli-responsive soft nanoparticles are classified depending on the stimulus (physical or chemical) into temperature, electric and magnetic field, light intensity, pressure, pH, ionic strength, specific (bio)molecules or enzymes and ultrasound-responsive nanoparticles. The development of soft stimulus-responsive nanoparticles for biomedical applications relies on the stimulus-sensitive polymer that constitutes their structure. Different polymer synthesis techniques are used and advanced polymerization processes have been developed to produce new stimuli-responsive nanoparticles, which are sensitive to other signals such as microwave, redox and different chemical substances, in addition to those already mentioned. Although the synthesis of new stimuli-responsive soft nanoparticles has attracted considerable interest in recent decades, in particular in the case of dual/multi-stimulus-responsive types, practical clinical applications remain limited except for thermo- and pH-sensitive nanoparticles with high sensitivities.

In recent years, several attempts to prepare thermo-responsive hybrid micro/nanogels with inorganic silica cores have been reported. These core–shell hybrid nanogels have interesting additional properties compared with polymer nanogels, being promising candidates as controlled drug-delivery vehicles in cancer therapies. From the synthetic point of view, in almost all studies the preparation of these nanohybrids consisted of three steps. First, the silica nanoparticles are prepared. The second step is the functionalization of silica with a coupling agent, mainly 3-(trimethoxysilyl) propylmethacrylate (TPM). This coupling agent generates a hydrophobic surface on the silica particles and in addition can subsequently react by radical polymerization, allowing chemical coupling between the polymer network and the inorganic material. The last step consists of the well-known emulsion polymerization used to synthesize nanogels. Ramos et al. reported a facile synthesis of thermo-responsive nanohybrids with a silica core and a poly(N-vinylcaprolactam) (PVCL)-based thermo-responsive shell. This was achieved by a batch emulsion polymerization of VCL with TPM and N,N'-methylenebisacrylamide (MBA) as cross-linkers. The hybrid character of these thermo-responsive nanogels together with the excellent biocompatibility conferred by silica and PVCL makes them suitable as carriers for drug delivery and biosensing.