Chapter 8: Nanomedicine via Freeze‐drying and Ice Templating – Ice Templating and Freeze-Drying for Porous Materials and Their Applications

Nanomedicine via Freeze‐drying and Ice Templating

8.1 Introduction

There have been continued efforts to develop new pharmaceutical formulations and novel delivery methods to improve drug bioavailability, therapeutic efficacy, and patient compliance whilst attempting to reduce the dosage and side effects. Nanomedicine and associated target and responsive delivery have been intensely investigated. In simple terms, ‘nanomedicine’ indicates the formulation and administering of active pharmaceutical ingredients (APIs) in the form of nanoparticles, usually in the size region of tens and hundreds of nanometres, be it drug nanocrystals, nanoencapsulation, nanoconjugation or with any form of nanocarriers [16]. A variety of methods have been developed and employed in nanomedicine research. In this chapter, the focus is on poorly water‐soluble drug nanoformulation to address poor solubility, poor bioavailability, and delivery of drug nanoparticles, particularly via the use of the freeze‐drying and ice‐templating methods.

8.2 Poorly Water‐soluble Drugs and Drug Classifications

More than 40% of drug compounds and new chemical entities (NCEs) in the pharmaceutical industry are poorly soluble in water [2, 7]. This situation has not been made less severe with the recently adopted high‐throughput synthesis and screening methodology. Poor water solubility leads to poor bioavailability, larger dosage, and hence severe side effects. As has been frequently quoted in literature, drug compounds may be termed as soluble or poorly water soluble. Poor water solubility may indicate a solubility less than ∼30 mg ml−1. Since water solubility is closely related to permeation and bioabsorption, these issues are often investigated or regulated together, for instance, by researchers in the pharmaceutical fields and regulation bodies such as the U.S. Food and Drug Administration (FDA). Drugs with regarding to solubility in water may be classified in more detailed categories. As shown in Table 8.1, the solubility categories include: very soluble, freely soluble, soluble, sparingly soluble, slightly soluble, very slightly soluble, and practically insoluble [8].

Table 8.1 Drug solubility classification in water.

Source: Adapted from

Solubility categorySolubility classificationSolubility range (mg ml−1)
Soluble in waterVery soluble>1000
Freely soluble100–1000
Poorly water solubleSparingly soluble10–33
Slightly soluble1–10
Very slightly soluble0.1–1
Practically insoluble<0.1

Biopharmaceutical classification systems (BCSs) have been proposed to facilitate drug development and approval by regulatory authorities. The BCS was introduced in 1996, based on solubility and permeability, to predict drug absorption [9]. The BCS classifies drug compounds into four categories: Class 1, Class 2, Class 3, and Class 4 (Figure 8.1). The criteria to determine whether a drug compound is highly soluble and/or highly permeable are also listed in Figure 8.1. The BCS system has been used by the U.S. FDA as a science‐based approach to allow waiver of in vivo bioequivalence studies for BCS Class 1 drugs (highly soluble and highly permeable) [10, 11]. As per FDA regulations, the solubility should be determined in aqueous media with a pH range of 1–7.5 by a shake‐flask or titration method, and analysed by a validated stability‐indicating assay. For the determination of dissolution rates, USP apparatus I (basket) at 100 rpm or USP apparatus II (paddle) at 50 rpm should be used in a defined medium (900 ml of 0.1 N HCl or simulated gastric acid, pH 4.5 buffer, and pH 6.8 buffer or simulated intestinal fluid). For the evaluation of intestinal permeability, both in vivo intestinal perfusion (in humans or animals) and in vitro permeation experiments can be performed. For in vitro permeability, several cell lines particularly Caco‐2 cell lines and Madin–Darby canine kidney (MDCK) cell lines have been widely used [6]. The in vivo permeability data from humans is always preferred because the in vitro data or in vivo data from animals may not produce the same results that can be applied in human medication.

Figure 8.1 The biopharmaceutics classification system (BCS) as proposed.

Source: Amidon et al. 1995 [9]. Reprinted with permission from Springer Nature.

There are 325 medicines and 260 drugs in the WHO (World Health Organization) Essential Drug List, of which 123 are oral drugs as immediate‐release products. These drugs have been classified based on the BCS system. Drug solubility is judged based on the dimensionless dose number, D0. D0 is the ratio of drug concentration in the administered volume (250 ml) to the saturation solubility of the drug in water. Drugs with D0 ≤ 1 are classified as highly soluble. The classification of permeability is based on log P or CLogP values. log P is the n‐octanol/water partition coefficient and can be calculated using three different fragmentation methods based on atomic contributions to lipophilicity. CLogP can be calculated using a program from BioByte Corp. log D, the pH‐dependent distribution coefficient for singly ionized species, can be calculated from log P and the ionization constant pKa. The drugs with estimated log P ≥ 1.72, CLogP ≥ 1.35, and log D ≥ −1.48 are classified as highly permeable. Based on dose number and log P, among the immediate‐release drug dosages, 23.6% are in Class 1, 17.1% in Class 2, 31.7% in Class 3, and 10.6% in Class 4 [8].

A drug compound is classified as highly permeable when the fraction of dose absorbed (Fabs) in humans is equal to or greater than 90%. However, there are reports that high Fabs may not simply translate to having high permeability, as shown by the high Fabs of the β‐blocker sotalol that has low Caco‐2 permeability [12]. This discrepancy may be attributed to the ‘local absorption’ or ‘average absorption’, with the latter usually used for Fabs. However, in the different sections of the small intestine, the environmental pHs are different, which can have a significant impact on the absorption of ionizable drugs. It can be emphasized that, if a compound has a high fraction of dose absorbed, it should show high permeability in the relevant intestinal regions [12].

However, with more data available on the increasing number of drug compounds, evidence has been growing that high extent of absorption does not necessarily indicate high permeability. Wu and Benet proposed the Biopharmaceutical Drug Disposition Classification System (BDDCS) based on solubility and metabolism (Figure 8.2) [13]. The BDDCS can be primarily used to predict drug disposition and the importance of transporters in drug absorption and elimination in the intestine and liver [10]. In the BDDCS, the major route of elimination for the highly permeable Class 1 and Class 2 drugs in humans is metabolism, whilst for the poorly permeable Class 3 and Class 4 drugs the main elimination route is via renal and biliary excretion of unchanged drugs[11]. Metabolism of ≥70% is set as the cut‐off value for extensive metabolism. It is noted that there are relatively few drugs where the extent of metabolism is in the range of 30–70%. Most drugs are either highly metabolized or poorly metabolized [11].

Figure 8.2 The biopharmaceutics drug disposition classification system (BCS) as proposed.

Source: Wu and Benet 2005 [13]. Reprinted with permission from Springer Nature.

Besides the drug classification systems, ‘the rule of 5’ proposed by Lipinski et al. has also been widely used to predict drug absorption or permeation. The ‘rule of 5’ suggests that a compound is more likely to exhibit poor absorption or permeation: (i) when the compound has more than five H‐bond donors (the sum of -OHs and -NHs); (ii) its molecular weight is over 500; (iii) its log P is over 5 (or MLogP is over 4.15); (iv) it has more than 10 H‐bond acceptors (the sum of -Ns and -Os); and (v) it is not a substrate for biological transporters [14]. Antibiotics, antifungals, vitamins and cardiac glycosides are exceptions to the ‘rule of 5’ because their structural features allow them to act as substrates for naturally occurring transporters. Although there are exceptions, the ‘rule of 5’ has been very effective and useful in drug development.

8.3 Nanoformulation Approaches for Poorly Soluble Drugs

A number of approaches have been developed to address the poor water solubility problem. Although it is always nice to start from drug design and then screen lead compounds and find soluble drugs with high therapeutic potent, this has always been extremely difficult to achieve, as evidenced by the poor solubility of potent drug compounds synthesized or discovered. In addition to this, synthesis of pro‐drugs is a useful route to enhancing solubility. Once absorbed, the pro‐drugs are metabolized and then released into the systemic circulation [2]. Crystal engineering, via the preparation of co‐crystals or by amorphous formulation and formation of salts, have also been exploited [1, 2].

However, nanosizing or nanoformulation techniques are probably most widely investigated and employed in research and development towards enhancing the drug solubility and bioavailability. The particle sizes can be reduced to microns and more favourably the nanosized range (e.g. 100–200 nm) [15]. The nanoparticles provide significantly increased surface area and also high surface energy. Both the dissolution rate and the saturation solubility can be improved as a result of nanosizing. The increase in dissolution rate can be described by Noyes–Whitney/Nernst–Brunner Equation [1517]:

where dM/dt is the dissolution rate by mass, A0 is the total surface area of the solute, D is the diffusion coefficient, h is the thickness of the boundary layer around the solute particle, cs is the saturation solubility and c is the solute concentration in the bulk phase at time t.

The increase in saturation solubility is given by the Ostwald–Freundlich equation [15, 17]:

where cs,r is the saturation solubility of a particle with radius r, cs,∞ is the saturation solubility of a particle with infinite radius, γ is the particle medium interfacial tension, M is the compound molecular weight, ρ is the particle density, R is the universal gas constant, and T is the absolute temperature.

The nanosizing techniques can be generally categorized into bottom‐up and top‐down methods. The bottom‐up methods start with drug solutions (mostly organic solutions due to their poor water solubility). The commonly used approaches include encapsulation (e.g. micelles, dendrimers, emulsions, nanocapsules, microspheres, and complexation) [2, 7, 18], solid dispersions (e.g. via hot melt extrusion) [7], nanoprecipitation, antisolvent precipitation (including the use of supercritical fluids and compressed fluids) [2], spray drying [19, 20], and cryogenic techniques (which will be the focus of this chapter) [2, 21].

There are not such a variety of top‐down approaches for nanosizing, with high‐pressure homogenization and ball milling (either dry milling or wet milling) being the mostly used techniques [15, 2224]. Owing to simple processing, easy control of process parameters, and scale‐up potential, the wet milling and homogenization methods are widely investigated and mostly used particularly in the pharmaceutical industry [1, 2, 15, 2124].

For nanosizing formulations, it is inevitable to use non‐aqueous solvents [25] and excipients [26, 27]. The solvent residual must be controlled within the limits in a drug product as required by regulatory bodies. The solvent may be categorized into three classes [28]. Class 1 solvents are human carcinogens, strongly suspected human carcinogens, or environmental hazards. These include benzene, carbon tetrachloride, 1,2‐dichloroethane, 1,1‐dichloroethene, and 1,1,1‐trichloroethane. These solvents should be avoided whenever possible. Class 2 solvents are non‐carcinogens and solvents suspected of other significant but reversible toxicities. Table 8.2 lists the Class 2 solvents for pharmaceuticals with permitted daily exposure (PDE) and concentration limits [28]. PDE is the maximum acceptable intake per day of residual solvent in pharmaceutical products. Class 3 solvents exhibit low toxic potential to human, with PDEs of 50 mg or more per day. The use of Class 3 solvents should be subject to good manufacturing practice (GMP) and other quality‐based requirements. Table 8.3 provides the list of Class 3 solvents [28]. There are also some solvents for which no adequate toxicological data are available: e.g. 1,1‐diethoxypropane, methylisopropyl ketone, 1,1‐dimethoxymethane, methyltetrahydrofuran, 2,2‐dimethoxypropane, petroleum ether, isooctane, trichloroacetic acid, isopropyl ether, and trifluoroacetic acid [28].

Table 8.2 Class 2 solvents (to be limited) for pharmaceutical products.

Source: Adapted from‐single/article/impurities‐guideline‐for‐residual‐solvents.html.

SolventPDE (mg d−1)Concentration limit (ppm)
Acetonitrile4.1               410
Chlorobenzene3.6               360
Chloroform0.6               60
Cumene0.7               70
Cyclohexane38.8               3880
1,2‐Dichloroethene18.7               1870
Dichloromethane6.0               600
1,2‐Dimethoxyethane1.0               100
N,N‐Dimethylacetamide10.9               1090
N,N‐Dimethylformamide8.8               880
1,4‐Dioxane3.8               380
2‐Ethoxyethanol1.6               160
Ethyleneglycol6.2               620
Formamide2.2               220
Hexane2.9               290
Methanol30.0               3000
2‐Methoxyethanol0.5               50
Methylbutyl ketone0.5               50
Methylcyclohexane11.8               1180
N‐Methylpyrrolidone5.3               530
Nitromethane0.5               50
Pyridine2.0               200
Sulfolane1.6               160
Tetrahydrofuran7.2               720
Tetralin1.0               100
Toluene8.9               890
1,1,2‐Trichloroethene0.8               80
Xylene21.7               2170

Table 8.3 Class 3 solvents (low toxic potential to human) for pharmaceutical products.

Acetic acidAcetoneAnisole
1‐Butanol2‐ButanolButyl acetate
tert‐Butylmethyl etherDimethyl sulfoxideEthanol
Ethyl acetateEthyl etherEthyl formate
Formic acidHeptaneIsobutyl acetate
Isopropyl acetateMethyl acetate3‐Methyl‐1‐butanol
Methylethyl ketone2‐Methyl‐1‐propanolPentane
Propyl acetateTriethylamine

Different excipients may be included in a pharmaceutical product to serve different purposes [26, 27]. The excipients participate in the systemic circulation like the APIs. Therefore, the safety of excipients is a very important issue but also highly complicated due to the variety of excipients used. Similarly to the APIs, the safety or toxicity of the excipients should be assessed [27]. Furthermore, the drug–excipient interaction [29] and the effect of the excipients on drug disposition [30] can have pronounced effects on excipient pharmacokinetics and profiling [31]. Sugars and polymers are the mostly used excipients for drug nanoformulations, either to protect the drug during processing or to stabilize the drug in the formulations [15, 19, 20, 26].

There are stability issues associated with nanosuspensions and solid nanoformulations. For nanosuspensions, the instabilities may include sedimentation/creaming, agglomeration, crystal growth, or crystallization of amorphous phase, and the potential chemical changes [32]. In solid formulations, the selection of excipients/bulking agents, the glass state of the matrix, and the molecular mobility (leading to nucleation/crystallization) of drugs in the matrix can have great impact on formulation stability [33].

Characterization of drug nanoparticles is an important aspect of drug nanoformulations [32]. Particle size, size distribution, and particle morphology can be obtained by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). It should be noted that these microscopic methods show the size and shape of dry nanoparticles although cryoTEM can be used to image the nanoparticles with stabilizer or hydration shell in a frozen state. Dynamic laser scattering (DLS) is an effective technique to measure the size of hydrated nanoparticles (but not particle shape) and surface charges. The data generated are very important for medical applications because the hydrated size of the nanoparticles (which can be considerably larger than the dry nanoparticles) is a good representation of the real size of nanoparticles after being administered. The crystalline state of the drug nanoparticles can be characterized by powder X‐ray diffraction (PXRD), wide angle X‐ray scattering (WAXS), small angle X‐ray analysis (SAXS), and differential scanning calorimetry (DSC) [22, 32].

Because one of the main purposes of employing nanosizing techniques is to enhance the drug solubility, the determination of dissolution rate and saturation stability of drug nanoparticles is crucial. It has been reported that the increase of saturation stability for drug nanoparticles is not more than 15%. Therefore, the enhancement in dissolution rate is the main contribution from nanoformulations [17]. Although the FDA‐recommended USP Apparatus 1 and Apparatus 2 can be employed to measure dissolution rate, questions have been asked on ‘what is an appropriate dissolution method for drug nanoparticles’. Because the drug nanoparticles are small, the nanosuspension is clear and it is difficult to judge a nanosuspension from a true molecular solution. Generally, the dissolution rates for drug nanoparticles are determined by separating the drug nanoparticle from the solution (e.g. by centrifugation and membrane dialysis) and the concentration of the solution can be determined by UV–Vis analysis or high performance liquid chromatography (HPLC). This measurement may be inaccurate, particularly when the separation time is long and the dissolution rate is fast. In situ analytical techniques such as UV–Vis fibre optic probe, calorimetry, and turbidimetry have been used, which obviate the need to separate dissolved API. However, the shortcomings of these methods are still related to the difficulty in distinguishing small nanoparticles and the molecular solution [17]. A recent development is the use of DLS to determine the dissolution rate and the solubility. This method is based on the linear correlation between intensity and particle number in diluted nanosuspensions. The variation of particle sizes must be also considered [17, 34].

In addition to the beneficial effect it provides of enhancing dissolution rate, aqueous nanoformulations may be administered both orally and intravenously [23, 24]. Bioabsorption, stability in systemic circulation, targeted delivery, and finally elimination of drug nanoparticles from the body are the essential benefits of administering pharmaceutical nanoparticles.

8.4 Bioavailability and Delivery of Drug Nanoparticles

Bioavailability may be defined as ‘the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of drug action’, as described in the Code of Federal Regulation, USA. Based on the Committee for proprietary medicinal products of the European Medicines Evaluation Agency (EMEA), bioavailability is understood to be the extent to and the rate by which a substance or its therapeutic moiety is delivered from a pharmaceutical form into the general circulation [35]. The bioavailability is achieved and governed by drug pharmacokinetics, which is the kinetics of drug absorption, distribution, metabolism, and excretion (ADME) [4, 31]. It is the same for drug nanoparticles, as shown in Figure 8.3. However, the nanoparticles can behave very differently at each stage of the ADME, compared to the dissolved drug molecules.

Figure 8.3 The schematic representation of the pharmacokinetic ADME for drug nanoparticles.

8.4.1 The Absorption Process

There are different administering routes, e.g. oral, intravenous, intramuscular, and pulmonary delivery. The oral administration is the most widely used, due to good patient compliance and no necessity for the presence of trained medical staff. The absorption process is involved in oral administration, pulmonary delivery, and skin delivery, etc. This section discusses oral administration and pulmonary delivery because of their wide use and because most of the processes may be adopted for other administration routes that involve absorption procedures. The Orally Administering Route

Figure 8.4 shows how the orally administered drug nanoparticles enter the blood systemic circulation. The absorption occurs mainly in the small intestine via membrane permeation. There are three sections in the small intestine, with different pH environment, which may have significant impact on the stability and absorption of ionized or pH‐sensitive drug compounds [12]. The duodenum is the shortest, widest, and the least mobile section, of 20–30 cm length and 3–5 cm diameter [36, 37]. It receives digestive enzymes from the pancreas and bile via the liver and gall bladder. This is where the degradation and/or dissolution mainly take place.

Figure 8.4 The absorption process for orally administered drug nanoparticles.

The wall of the small intestines is composed of mucosa, submucosa, muscle layers, and serosa. The serosa consists of a single layer of flattened mesothelial cells overlying some loose connective tissues. The submucosa is a network of connective tissues containing small blood vessels, lymphatic, and nerve plexus. The mucosa is at the inner surface of the intestine and is directly in contact with the intestinal fluid. The epithelium is the innermost layer facing the lumen of the bowels, consisting of a single layer of columnar epithelial cells (enterocytes), which line both the crypts and villi [37]. The presence of villi and microvilli, mostly present in the duodenum and jejunum, provides the large absorption surface area. For this reason, the small intestine is considered to be the major absorption site [38].

The small intestine lies anterior to the liver. The blood flows from the small intestine immediately into the liver by way of the portal vein. In the liver, the blood passes evenly through the hepatocytes and ultimately leaves the liver via the hepatic veins that empty into the vena cava of the general circulation. The amount of orally administered drugs can be reduced by both intestinal and hepatic metabolism. This effect is referred as first‐pass metabolism [37] (Figure 8.3). The first‐pass metabolism includes only the metabolism by the intestine during absorption and by the liver when the blood flows immediately into it from small intestine [38]. The bioavailability (F) by oral administration can then be described as [39]:

where fabs is the fraction of the dose absorbed from the intestinal lumen, fi is the fraction of drug metabolized by the small intestine, and fh is the fraction of the drug metabolized by the liver.

During the absorption by the small intestine, the drug compounds or nanoparticles must first overcome the mucousal barriers. Mucousal surface protects all major portals of the body such as nose, lung, intestine, gall bladder, and reproductive tracts, by excluding pathogens and other dangerous materials. The mucousal surface is negatively charged due to the presence of sialic acids and sulfated monosaccharides in the sugar chains. The thickness of the mucous layer varies at the different parts of the body, about 300 μm in the stomach and 700 μm in the intestine [40]. As mentioned earlier, the epithelium layer lines the mucousal surface, with the two most abundant epithelial cells being absorptive cells (enterocytes) and secretory cells (goblet and Paneth). Drug nanoparticles may be predominantly taken by the M cells in the Peyer's patches of the ileum. The size of the nanoparticles can make a difference to the way the nanoparticles are absorbed. Nanoparticles with sizes less than 50 nm are transported paracellularly, whilst the nanoparticles of 50–200 nm are endocytosed by enterocytes and those of 200 nm to 5 μm are taken up by M cells of the Peyer's patches [41, 42].

The mechanism of the drug permeating across the epithelium layer can be transcellular and paracellular, as shown in Figure 8.5. Sometimes, unexpected bioavailability characteristics are observed, either higher or lower than expected. This can be attributed to the influx and efflux processes facilitated by transporter proteins, which include passive transporter (moving solutes from high concentration to low concentration or via an electrochemical gradient) and active transporter (moving solutes against the concentration or electrochemical gradient). An energy‐yielding chemical or photochemical reaction is required to facilitate the active transporter. P‐glycoproteins can effectively utilize the ATP‐produced energy to facilitate the active efflux of its substrates [39].

Figure 8.5 The schematic representation for the permeation pathways across the intestinal membrane.

Source: Löbenberg and Amidon 2000 [35]. Reprinted with permission from Elsevier.

For the transcellular permeation, the drug molecules or nanoparticles move across the lipid bilayers of epithelial cells. The physicochemical properties of the compound and the structure of the lipid bilayers can exert significant impact on the permeation. In general, the bilayer can be divided into four distinct regions: (i) the first, the outermost hydrophilic region containing a high proportion of water molecules; (ii) the second, the highly packed dense region of polar heads, acting as the greatest barrier to solute diffusion; (iii) the third, the hydrophobic region containing the highest density of nonpolar tails; and (iv) the fourth, the most hydrophobic, region of the membrane, serving as a hydrophobic barrier [39]. It is clear that the lipid bilayer is a complex structure of hydrophilic and hydrophobic regions. Both highly hydrophobic and highly hydrophilic compounds lead to poor permeation. Indeed, it is suggested that the optimal partition coefficient log P0 (based on partitioning between n‐octanol and water) for a drug is in the region of 2–7 [39] while others suggest that drugs with log P0 in the range of 1–3 show good passive absorption across the lipid membrane [6].

During the absorption process in small intestines, blood flow in the villi as well as the adjacent regions of the submucosa is increased greatly. Drugs permeate across the villi and into the blood and then into the liver, followed by systemic circulation. The first‐pass metabolism occurs in both the small intestine and the liver. Cytochromes P‐450 are the dominant enzymes involved in the biotransformation of drugs. The three main P‐450 gene families, CYP1, CYP2, and CYP3 are thought to be responsible for drug metabolism [37]. The distribution of P‐450 in the small intestine is not uniform, with the content higher in the proximal than in the distal part. The average total cytochrome P‐450 content in human intestine is about 20 pmol mg−1 microsomal protein, much lower than that in liver (about 300 pmol mg−1 microsomal protein). It is thus believed that the liver contributes significantly more to the first‐pass effect. It has been found that CYP3A4 is the dominant cytochrome P‐450 type in the mucousal epithelium in human small intestine [37]. The drug metabolism is usually considered to be in two phases. The Phase 1 metabolism generally degrades or modifies the drug compounds via the reactions of hydrolysis, oxidation, and reduction. In the Phase 2 metabolism, the drug compound or its metabolites are conjugated to certain functional groups to make them water‐soluble and be easily excreted [38].

Various methods may be employed to investigate drug absorption or first‐pass metabolism in the small intestine. These include the in vitro method (e.g. with tissue slices, Caco‐2 cells/Goblet cells, rat intestine), the in situ method (vascular perfusion), and the in vivo method (e.g. perfused intestinal loops, portacaval transposition, AUC (area under curve) measurement) [38].

Despite different permeation pathways (either transcellular or paracellular), the overall absorption rates of most drugs may be commonly described by Fick's First Law, where the mass transport across the membrane is proportional to the concentration difference and the absorption area and also determined by the diffusion coefficient [35, 37]. The blood flow close to the epithelium layer can be treated as unstirred water. A high drug solubility or high concentration of nanosuspension in the intestine lumen can lead to fast absorption. A balance of hydrophobicity and hydrophilicity of the drug, allowing adequate solubility in aqueous luminal fluid and lipid membrane phase of the enterocytes, is highly important to achieve optimal intestinal absorption. Absorption in Lung by Pulmonary Delivery

Similar to the intestinal tract, the lung is in direct contact with the environment and can act as the first port of entry for nanoparticles into the body. The lung consists of the airways (trachea, bronchi, and bronchioles) and the alveoli (gas exchange areas). The respiratory zone includes all the structures that participate in gas exchange, beginning with the bronchioles. The gas exchange mainly occurs in the alveolar region due to the high surface area. The surface area of the human lungs is estimated to be about 75–140 m2 in adults [43]. The mucus lines the respiratory epithelium from the nose to the terminal bronchioles. In humans, the mucous layer consists of the periciliary (sol) layer (∼ 5–10 μm thick) and the luminal (gel) layer (∼60 μm thick). The sol layer is close to the epithelium and more watery. The gel layer contains cilia to propel mucus up and out of the lung, acting to remove external particles [40]. The pseudostratified epithelia are different in airways and alveoli of the lungs, with a gradually thinning columnar epithelium in airways, 3–5 mm thick in the bronchial epithelium and 0.5–1 mm thick in the bronchiolar epithelium. The particles deposited in this region are either moved away from the lung by mucociliary clearance or diffuse through the thick mucus to the epithelial cells. However, in the alveolar region, there exists a thin, single cell layer. The distance between the air in the alveolar lumen and the capillary blood flow is less than 400 nm. As such, the build‐up of nanoparticles in the alveolar region poses great health risks [43].

The deposition of particles in the respiratory tract can be described by three main mechanisms. The first mechanism, inertial impaction, occurs during the passage through the oropharynx and large conducting airways for the particles larger than 5 μm (based on the mass median aerodynamic diameter, MMMD). The second one, gravitational sedimentation, is responsible for the particles of 1–5 μm in smaller airways and respiratory bronchioles. The third one, the Brownian diffusion, is for particles less than 0.5 μm. The main factors impacting on particle deposition include: (i) particle size, density, shape, and surface properties; (ii) the structure of airways and alveoli; and (iii) ventilator parameters such as breathing pattern, flow rates, tidal volume, etc. Figure 8.6 shows the effect of particle sizes on the deposition in different parts of the human respiratory tract [44]. The optimal particle sizes to achieve delivery deep in the alveolar region are in the range of 1–3 μm. Large particles tend to deposit in the mouth and throat and airways. Smaller particles can increase both deposition (the ventilation parameters should be considered for the highly mobile and suspended nanoparticles) and diffusion in the alveolar region.

Figure 8.6 The effect of particle size on the deposition of aerosol particles in the human respiratory tract following a slow inhalation and a 5 s breath hold. Larger particles deposit in the airways or mouth and throat, whereas smaller particles deposit in the alveolar region. The optimal size range for deposition in the alveolar region is 1–3 μm.

Source: Bryon 1986 [44]. Reprinted with permission from Elsevier.

The discussion on the nanoparticle penetration across the epithelium of skin, nose, and vagina can be found elsewhere [40].

8.4.2 Nanoparticle Clearance and Distribution

Once the drug nanoparticles enter the systemic circulation, either by oral administration (with first‐pass metabolism) or other routes (e.g. intravenous injection, or pulmonary delivery), the nanoparticles may be eliminated by the reticuloendothelial system (RES) (Figure 8.3). However, for the drug nanoparticles to generate therapeutic efficacy, sufficiently long circulation time and transport to the target tissue cells are required.

The RES, also known as mononuclear phagocyte system (MPS), is part of the immune system that consists of phagocytic cells, e.g. monocytes in blood and macrophages in tissues such as liver, spleen, and bone marrow. Phagocytes have receptors for molecules, termed as opsonins, which initiate binding and removal [45]. There are various proteins and ions in the blood, with human serum albumin being the most abundant protein. The absorption/binding of proteins and/or ions may cause nanoparticle aggregation and precipitation. More importantly, this can initiate nanoparticle opsonization. This process tags the nanoparticles, which can be subsequently recognized and taken up by phagocytic cells. This is an effective way to remove foreign particles from the body, mainly by the hepatic Kupffer cells and the marginal zone and red‐pulp macrophages in the spleens [4, 45]. The hepatic Kupffer cells have special features (ciliated borders and stellate branches) that facilitate the removal of foreign nanoparticles. Kupffer cells contain various receptors for selective endocytosis of opsonized foreign particles. Hepatocytes contribute considerably to endocytosis and enzymatic breakdown of foreign particles. The nanoparticles that cannot be broken down by intracellular processes remain in the macrophages and hence in the body [46]. As a function to protect the body, the liver can efficiently capture and eliminate nanoparticles in the range of 10–20 nm (including viruses) [46].

In order to prolong the systemic circulation in blood, the nanoparticles can be surface‐modified with stealth‐shielding coating. Different polymers such as polysaccharides, polyacrylamide (PAM), and poly(vinyl alcohol) (PVA) have been used to modify the particle surface [47]. However, coating the nanoparticles with poly(ethylene glycol) (PEG) or PEG‐copolymer have proven to be mostly successful [47, 48]. The protective action of PEG is mainly attributed to the hydrophilic shell on the particle surface. The water molecules can form a dense shell via hydrogen bonding to the ether oxygen of PEG molecules. This hydrated surface shell can repel the protein and reduce the interaction. It is also suggested that the PEG chains can undergo spatial conformations and prevent the opsonization of particles by the macrophages of the RES [47]. Investigation on the novel coating on nanoparticles to facilitate negligible immune reactions is an exciting and emerging research area for drug delivery [48].

The transport path of the nanoparticles from blood circulation to the target cell and its nucleus is schematically shown in Figure 8.7. The nanoparticles need to permeate across the endothelium, diffuse through tissue interstitium, and arrive at the cell surface. Via endocytosis, the nanoparticles can move across the cell membrane and diffuse into different parts including the nucleus inside the cell. As described in Figure 8.7, there are different types of endothelium and different sizes of transcellular channels or fenestrations at different parts of the body [40]. In addition to particle sizes, the shape of the particles plays a significant role in the transport across the endothelium. For example, it has been reported that the cellular internalization (with the HeLa cells) of rod‐shaped, high‐aspect‐ratio nanoparticles occurred more easily than that of the more symmetrical and low‐aspect‐ratio particles [49]. The particle size, shape, and surface chemistry can all play significant roles in cellular internalization, as evidenced in the study with gold nanorods [50]. There are two exceptional examples of endothelium. The first is the blood–brain barrier (BBB), the tightest endothelium in the body. Discovering drugs and/or functionalized nanoparticles to move across the BBB have always been a great challenge. The second one is the leaky vasculature around tumour cells, which allows the passive permeation of large nanoparticles, widely known as the enhanced permeability and retention (EPR) effect [40, 45]. The growing solid tumour requires nutrients and metabolites for growth. The resulting neovasculature is not hierarchically organized as in capillary beds. This heterogeneity can lead to variable accumulation of drug nanoparticles [45]. The pore sizes in the leaky tumour vasculatures are reported to be in the range of 380–780 nm [40] while others suggest that the highly heterogeneous cut‐off pore sizes are in a wider range of 200–1200 nm [51]. Different factors can influence the transport of drug nanoparticles from blood to tumour cells. The chaotic blood flow in the tumour vasculature reduces the particle extravasation into the tumour interstitium. The higher interstitium pressure also hinders the transport into the interstitium.

Figure 8.7 The scheme describes the delivery of drug nanoparticles from blood circulation to the target cell.

Source: Adapted from Refs. [40, 52].

As noted in Figure 8.7, there is variation of pHs in tumour tissue interstitium and tissue cells. Functional coating on the drug nanoparticles can facilitate the targeted delivery and the responsive release at the target sites. The coatings (either chemically bound or physically attached) can provide stealth shielding [47] and also prevent the rapid dissolution of drug nanoparticles before reaching the target. In addition to the long circulating polymer coating, the target‐binding ligand and/or polymers with responsive bonding or functional groups can be further attached to provide multifunctional and responsive drug nanoparticles [52]. Some examples include PEG linkage with folate/peptide/nanoplex [47], pH‐sensitive coating with zwitterionic chitosan derivatives [53], intracellular reduction‐sensitive release [54], and tumour intracellular pH responsive delivery [55].

8.4.3 Metabolism and Excretion

Once the therapeutic roles are achieved, the drug nanoparticles should be removed from the body, by metabolism and/or direct excretion via renal clearance (Figure 8.8). As many nanoparticles (e.g. quantum dots, Au nanoparticles) may not be metabolized, to avoid taking up by macrophages and accumulating in the body, excretion via urine through kidney processing is the major route. The process involves glomerular filtration, tubular secretion, and then elimination through urinary excretion. The nanoparticles in systemic circulation enter the glomerular capillary bed via the afferent arterioles. The glomerular wall consists of fenestrated endothelium, the highly negatively charged glomerular basement membrane (GBA) and the foot processes of glomerular epithelial cells (podocytes), which are separated by filtration slits bridged by slit diaphragms. The sizes of the slit diaphragms are around 43 nm [56]. However, the physiological pore size is much smaller, only in the diameter range of 4.5–5 nm. This means that the nanoparticles less than 5 nm can be easily excreted by glomerular filtration. For the nanoparticles in the range of 6–8 nm, the glomerular filtration depends on the shape and surface charge of the nanoparticles. Owing to the negative charge of the GBA, the positively charged nanoparticles can be easily excreted [46]. However, for larger nanoparticles that do not undergo glomerular filtration, they continue to be in the circulation and may be removed by the hepatic clearance (or building up in the body) via macrophage uptake (Figure 8.8).

Figure 8.8 The scheme shows the fate of drug nanoparticles in the body.

Particle shapes can have significant impact on renal clearance. The nanotubes or nanorods with high‐aspect‐ratios may be rapidly removed by glomerular filtration. For example, the single‐walled carbon nanotubes with average lengths of 200–300 nm could undergo rapid renal clearance. This was facilitated by the flow‐induced orientation of the nanotubes [57]. However, for the size‐controlled spherical quantum dots, efficient urinary excretion in rodents was demonstrated only for the particles with hydrodynamic diameters of ≤5.5 nm [58].

For pharmaceutical nanoformulations, the addition of excipients and/or stabilizers is unavoidable. For oral administration, the drugs need to have a balanced lipophilicity–hydrophilicity to be absorbed across the intestinal membrane. The hydrophilic excipients may be chosen so that they may have limited permeation across the biological membrane barrier, although many excipients are lipophilic [31]. Hydrophilic excipients may be easily excreted via glomerular filtration. The hydrophilic excipients (e.g. polyvinylpyrrolidone (PVP), PVA, and PEG) with molecular weights less than 10 kDa can be readily removed from the body. The elimination t1/2 (the time required to reduce the plasma concentration by 50%) increases with the excipient molecular weight in a sigmoidal curve shape [31].

8.4.4 Nanoparticle Toxicology

Owing to the increasing use of nanotechnology, the risks associated with the exposure to nanoparticles should be thoroughly evaluated. However, the toxicological effects of nanomaterials and the mechanisms are not well understood and indeed have not received sufficient attention. The same characteristics of nanoparticles that are attractive in nanomedicine may contribute to the toxicological consequence in biological systems.

The routes of entry into the body by nanoparticles include the respiratory system (the most important one), the skin (via exposure, cosmetics), and the gastrointestinal (GI) tract (via food, drink) [59]. The widely known example is the adverse health effect from exposure to asbestos fibres [60]. The airborne particles in air pollution are a global threat to human health, particularly in developing countries where the pollution‐heavy industries are not well regulated and monitored. A typical ambient particulate matter (PM) is a highly complex mixture of particles with median diameters in the range of low nanometres to 100 μm. The fraction or concentration of the particles with median diameter of 2.5 μm or less (known as PM2.5, widely mentioned in monitoring air quality) can deposit deep into the lung and cause substantial damage [43, 44]. Inhalation of PM2.5 and ultrafine particles (0.1–2.5 μm) can cause inflammatory and granulomatous responses in lungs, lung injury via oxidative stress, and cardiovascular dysfunctions [59].

After entry into the body, the nanoparticles can diffuse/permeate across the biological membrane and into the systemic circulation. The modes of permeation and diffusion can be very similar as those of drug nanoparticles, as described in the earlier sections. The difference, however, is that these nanoparticles do not have any intended therapeutic roles but can cause toxicological consequences if they cannot be removed from the body by the RES clearance [24].

In conventional toxicology, the key parameters are concentration and time. However, for nanoparticles, the dose metric is more related to the number of the nanoparticles arriving at the site of the target organs or tissues. The increased surface area of nanoparticles contributes to the increasing surface adsorption and surface reactivity. The key parameters of the nanoparticles that influence the toxicology are the size, shape, and surface chemistry of the nanoparticles, and also the nature of the nanoparticle substance. The interaction mechanism at the nanoparticle–biological interface may be classified as chemical or physical [61]. Of the chemical mechanisms, the generation of reactive oxygen species (ROS), considered to be the main chemical process in nanotoxicology, can lead to inflammation, cell damage and even cell death [62]. The physical mechanisms mainly include the disruption of membrane, transport process, protein confirmation/folding, and protein aggregation/fibrillation [63]. Once engulfed into the cell, the nanoparticles can interfere with cell signalling process, interact with proteins by chaperone‐like activity or inducing peptide aggregation and fibrillation. However, the potential damage to DNA is not well understood but may be explained by the various chemical and physical mechanisms (particularly by the ROS route) [63]. Overall, the investigation into the toxicological impact of nanomaterials to human health (and the environment) is still at the early stage. Understanding of the entry of the nanoparticles into the body as well as of the mechanisms of interaction with the cells in various biological environments is required so that, in addition to formulation of regulation policies, proper procedures for protection may be developed.

8.5 Freeze‐drying of Solutions/Suspensions for Nanomedicine

Freeze‐drying of aqueous solutions to prepare solid biopharmaceutical formulations (mostly proteins based) is the most investigated and applied area [64]. Because freeze‐drying is an energy‐intensive and time‐consuming technique, any modifications leading to faster freeze‐drying are preferable. Thus, instead of generating frozen monoliths, a spray freeze‐drying process can be employed, where a solution or suspension is atomized into cold vapour over a cold liquid (usually liquid nitrogen) or directly atomized into liquid nitrogen (readers may refer to Figure 3.1 for the process scheme in Chapter 3) [65]. Further developments include thin film freeze‐drying or spin coating freeze‐drying [66].

When employing freeze‐drying for poorly water‐soluble drugs, the main purpose is to reduce particle size and enhance drug dissolving rate and bioavailability. However, due to the poor water solubility, non‐aqueous solvents are often added to facilitate the wettability and produce a mix solution [25]. A range of organic solvents have been used, which are usually miscible with water. These non‐aqueous solvents usually have higher vapour pressure than water in the frozen state, which facilitates fast sublimation during primary drying. The mostly used co‐solvent is tert‐butanol (freezing point 24 °C, vapour pressure 26.8 mmHg at 20 °C). The co‐solvents with higher freezing points are usually preferred because this raises the glass transition temperature of the frozen solids. In contrast, for the co‐solvents with low freezing points (e.g. ethanol, acetone, ethylacetate), a low percentage (e.g. <10%) may be only used. Otherwise, the liquid formulations may not be frozen in a commercial freeze‐dryer or the thawing of frozen samples may occur during freeze‐drying. Other suitable solvents may include dimethylcarbonate (freezing point 2 °C, vapour pressure 72 mmHg at 20 °C, but limited solubility in water) or dimethylsulfoxide (freezing point 18.4 °C, vapour pressure 0.5 mmHg at 20 °C, miscible with water) [25]. In addition to the physicochemical properties, the toxicity of the solvent and the solvent residual in pharmaceutical products should be considered. As listed in Tables 8.2 and 8.3, the use of Class 2 solvents should be limited and the solvent residual must be controlled within the limit.

The BCS Class II drug celecoxib was dissolved with Phospholipid E80 and trehalose (optimal ratio 1 : 10 : 16) in tert‐butanol‐water (60/40 w/w). The solution was frozen at −80 °C for 24 h and then placed in a pre‐cooled freeze‐dryer at −60 °C. The freeze‐dried formulation showed enhanced dissolution rate, apparent solubility, and concentration in phosphate buffer solution (pH 6.5) [67]. Danazol was dissolved in tetrahydrofuran (THF) and then mixed with aqueous solution containing PVA (Mw = 22 K) and PVP K‐15. A spray freezing was applied to liquid nitrogen process to produce frozen micron powders, which were subjected to vacuum freeze‐drying, or atmospheric freeze‐drying [68].

Organic solvents are used to dissolve the poorly water‐soluble drugs. For example, danazol and PVP K‐15 were dissolved in acetonitrile or acetonitrile/dichloromethane. The organic solutions were atomized into small droplets directly into liquid nitrogen. This processing led to the dissolution of 95% danazol in 2 min compared to 30% of the bulk danazol dissolved in 2 min, determined using USP Apparatus 2 under sink conditions [69].

However, due to the concerning issues on the toxicity, safety, and environmental impact of the organic solvents, much effort has been directed to the use of an aqueous medium. For poorly water‐soluble drugs, additional procedures are required to generate the suspensions of hydrophobic drugs, which are then freeze‐dried, mainly for storage stability, facile handling, and transport. For example, the evaporative precipitation into aqueous solution was employed to make the BCS Class II drug (danazol, itraconazole, carbamazepine) suspensions that were then processed by spray‐freeze‐drying. The resulting amorphous drug particles showed increased dissolution rate and increased drug bioavailability and efficacy [70]. Other approaches before freeze‐drying include micellar encapsulation of hydrophobic drugs [71, 72], drug encapsulation by nanoparticles [73, 74], and nanocomplex formation of cationic and anionic species [75, 76].

Freeze‐drying or spray‐freeze‐drying of nanoparticle suspensions are not limited to poorly water‐soluble drug nanoparticle suspensions. These techniques are also widely used for aqueous nanoparticle suspensions containing proteins, peptides, oligonucleotides, and DNAs [7781]. The main targets are to maintain pharmaceutical stability during storage, transport, and after reconstitution. Lyoprotectants and stabilizers are required to achieve these targets [82, 83]. More information on this topic can be found in Chapter 3.

8.6 Emulsion‐Freeze‐drying for Drug Nanoparticles

Emulsions are formed when mixing hydrophobic organic phase (water immiscible) and aqueous phase in the presence of a surfactant (or stabilizer) usually by stirring or homogenization, with the internal droplet phase dispersed in the continuous phase. Although there are emulsions formed from two immiscible non‐aqueous phases, the cases are rare. The emulsions can be categorized as oil‐in‐water (O/W) emulsions (where the oil phase is dispersed as droplets in the continuous aqueous phase) or vice versa water‐in‐oil (W/O) emulsions [84, 85]. For hydrophobic drugs and bioactives, the O/W emulsion is an effective tool for their dispersion, absorption, and enhanced bioavailability [8488]. Particularly for drug delivery, lipid‐based emulsions have been mostly used, either for oral or parenteral administrations. Lipids are broadly defined as biological substances that are hydrophobic and usually insoluble in water [86, 87]. Lipids may be divided into ‘simple’ lipids (where the hydrolysis reaction produces two products at most) and ‘complex’ lipids (where three or more products are formed upon hydrolysis). To facilitate the communication in the international lipids community and be compatible with informatics and lipidomics requirement, Fahy et al. divide lipids into eight categories (fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) [89]. Fatty acids, glycerolipids, and glycerophospholipids are mostly employed in lipid‐based emulsions and nanospheres. Among them, medium‐chain glycerides (MCTs) with 6–10 carbons (one type of glycerolipids) are widely used as the internal oil phase for the lipid‐based emulsions. Some natural sources, e.g. coconut oil and diary fat, exhibit similar properties as MCTs [90].

Based on the size of the droplets, emulsions can be categorized into emulsions (droplet sizes from sub‐micron to 100 μm, thermodynamically unstable), nanoemulsions (droplet sizes approximately 50–500 nm, better kinetic stability), and microemulsions (clear and thermodynamically stable, droplets <100 nm in diameter) [91]. All these types of emulsions have been used for drug delivery. However, the stability of the emulsions (even for the thermodynamically stable microemulsions, but may be kinetically unstable) and chemical stability of the drugs (e.g. hydrolysis in the presence of water) are important issues that need to be addressed [91]. Thus, emulsions are freeze‐dried to improve the stability and/or generate porous dry tablets. Most of the reported studies have focused on the stability test and/or enhanced dissolution rate [9297]. The emulsion with MCTs (the commercial Miglyol 810/812) as internal oil phase was freeze‐dried to produce porous tablet [96, 97]. However, considering the very low vapour pressure of the MCTs (e.g. vapour pressure < 0.0075 mmHg at 20 °C for Miglyol 812, based on the Material Safety Data Sheet from the Caesar & Loretz GmbH), it would be difficult to remove the MCT by freeze‐drying. However, most of the papers did not comment on what happened to MCT during freeze‐drying and there have been no reports (to the best of the author's knowledge) on the size of drug particles from this emulsion‐freeze‐drying approach.

Freeze‐drying has been an effective route to the preparation of a variety of porous materials [98]. When emulsions are freeze‐dried, porous materials templated by both droplets and ice crystals can be produced [84, 98]. When a polymer is dissolved in the droplet phase, porous microparticles or microcapsules can be prepared by emulsion‐freeze‐drying [99, 100]. However, when small molecular hydrophobic compounds are dissolved in the droplets of O/W emulsions, hydrophobic organic nanoparticles (including poorly water‐soluble drug nanoparticles) can be readily formed [2, 101]. The concept for the preparation of organic nanoparticles was initially demonstrated by crosslinking polymerization of an O/W emulsion followed by freeze‐drying (Figure 8.9) [102]. A hydrophobic dye, Oil Red O (OR), was used as the model hydrophobic compound, because it was very easy to judge from the colour/transparency of the suspension whether aqueous organic nanosuspension was formed. The emulsion‐freeze‐drying method was then developed as a highly efficient method to produce solid drug nanoparticles and highly stable aqueous nanoparticle suspensions. The OR nanoparticles could be released linearly in hydrophilic crosslinked polymers such as PAM or in a burst fashion in response to temperature change when poly(N‐isopropyl acrylamide) (PNIPAM) was the polymer matrix (Figure 8.9e). Similarly, the reduction‐controlled release of organic nanoparticles and the temperature‐responsive pumping release from PNIPAM have also been demonstrated [103, 104].

Figure 8.9 The schematic representation of formation of organic nanoparticles by emulsion templating and freeze‐drying and the release of organic nanoparticles. (a) An O/W emulsion is formed with a hydrophobic dye (Oil Red O) in the internal droplet phase and monomer/initiator in the continuous aqueous phase. (b) After polymerizing the emulsions followed by freeze‐drying, organic nanoparticles are formed in the porous polymeric matrix. (c) For temperature‐sensitive polymer PNIPAM, when the temperature is less than the low critical solution temperature (LCST), the nanoparticles remain inside. (d) When the surrounding temperature is greater than the LCST, the organic nanoparticles are squeezed out into the aqueous medium. (e) The plot shows the linear release of organic nanoparticles for the hydrophilic polymer polyacrylamide (PAM) and the burse release in response to the temperature for PNIPAM.

Source: Zhang and Cooper 2007 [102]. Reprinted with permission from John Wiley and Sons.

However, the organic nanoparticles within crosslinked porous polymers [102104] may not be a good choice for the production of poorly water‐soluble drug nanoparticles because the dominant administering routes have been oral and parenteral. In both cases, a soluble matrix or an aqueous nanoparticle suspension is required. Thus, instead of polymerizing an emulsion, hydrophilic polymers (e.g. PVA) may be added to the continuous phase of an O/W emulsion. Figure 8.10 shows the result of freeze‐drying the O/W emulsion where OR was dissolved in the internal cyclohexane phase and PVA and anionic surfactant sodium dodecyl sulfate (SDS) in the continuous water phase [105]. This is a physical process with no chemical reactions involved. The formed emulsion was injected into liquid nitrogen and the frozen beads were then freeze‐dried to produce the dry red beads. When water is added, the red beads can be dissolved in water rapidly, producing a clear red suspension, which looks no different from OR‐acetone solution. However, when the as‐purchased OR is added to water, it only floats on the surface (Figure 8.10a–c). This is an indication that the OR nanoparticles are formed and well dispersed in water. Characterizing the dry beads (Figure 8.10d) shows the highly interconnected porous structure (Figure 8.10e), which contributes to the fast and instant dissolution of the red beads. Some of the nanoparticles can be seen on the pore wall by SEM imaging of the dry bead (Figure 8.10f). The OR nanosuspension was further characterized by SEM (by depositing a drop of the suspension on a copper grid and then allowing to dry) and by DLS, which shows the size of OR nanoparticles around 90 nm (Figure 8.10g,h) [105]. As a demonstration, the poorly water‐soluble triclosan nanoparticles were prepared by this method. For the triclosan solution in ethanol/water, the observed MIC50 in inhibiting the bacterial (Corynebacterium) growth was approximately 50 ppm. In contrast, the aqueous triclosan nanodispersion was considerably more active, with the bacterial regrowth inhibited to <50% at 6.25 ppm [105].

Figure 8.10 Characterization of solid nanocomposites and nanodispersions. (a–c) Visual comparison of aqueous nanodispersion of Oil Red (OR) (a), OR powder added to water (b), and OR dissolved in acetone (c). (d) Highly porous nanocomposite beads containing OR. (e) SEM image of the internal porous bead structure. (f) Higher magnification showing nanoparticles on the edge of a pore. (g) STEM image of OR nanoparticles after drying the nanodispersion onto a porous carbon grid. (h) Dynamic light scattering analysis of the OR nanodispersion showing an average particle size of 90 nm.

Source: Zhang et al. 2008 [105]. Reprinted with permission from Springer Nature.

This method can be readily applied to other hydrophobic dyes and poorly water‐soluble drugs [106115]. Compared to the drug nanoparticles prepared by conventional methods such as wet milling and homogenization, the emulsion‐freeze‐drying method can be applied to compounds of hard and soft, heat‐sensitive or pressure‐sensitive compounds, which can avoid the impurities introduced by ball milling. Importantly, the method can produce nanoparticles below 300 nm (which is considered to be the cut‐off size for stable nanoparticle suspensions [1]) and prevent nanoparticle aggregation because they are stabilized in a porous solid matrix. The highly interconnected porous nature of the hydrophilic matrix allows the instant dissolution of the matrix to generate stable aqueous nanoparticle suspensions for further evaluation and application. One of the advantages with emulsions is that the volume percentage of internal phase can be varied in a wide range. When the volume percentage of the internal phase is higher than 74.05%, where the droplets are tightly packed together, the emulsion is called a high internal phase emulsion (HIPE). HIPEs can be employed as templates to produce highly interconnected porous materials [84]. When the volume percentage of the droplet phase is varied (e.g. from 20% to 80%) in an emulsion, the porosity and morphology of the resulting materials can be systematically tuned [107]. This approach may be used to effectively tune the loading of the drug nanoparticles in a formulation. The size of the nanoparticles (e.g. indomethacin) may be controlled by the concentration of the internal organic phase [108].

For the emulsion‐freeze‐drying approach, surfactants are required to stabilize the emulsion while a hydrophilic polymer is needed to form the porous matrix to hold the nanoparticles. In the solid state, the organic nanoparticles can be very stable. Some samples have been stored over 1 year and can still produce transparent nanoparticle suspensions upon dissolution. Both the polymer and surfactant may be treated as excipients for pharmaceutical formulations. Thus the toxicity and biocompatibility of the surfactant and polymer must be considered. Some surfactants may be fine for oral administration but are not ideal for intravenous injection. With this in mind, from the point view of chemistry and materials, designing and synthesis of functional polymers to act as both stabilizers and bulking polymers should be crucial. Recently, a lightly crosslinked branched block copolymer PEG‐b‐PNIPAM was synthesized and used to form O/W emulsion. The polymer was present in water as core–shell nanoparticles with lightly crosslinked hydrophobic core. The emulsion‐freezing approach was applied to form highly porous polymer–nanoparticles composite, which can be readily dissolved to generate stable aqueous drug nanoparticle suspensions, with the nanoparticle yields achieving as high as 100% for ketoprofen and ibuprofen [109]. With different design and synthesis, for example, with a more hydrophobic core of poly(butyl methacrylate), there may be great potential to identify suitable functional polymers for targeted drug nanoparticles [110].

With the emulsion‐freeze‐drying approach, it is easy to prepare multicomponent organic nanoparticles. This can be simply achieved by dissolving or dispersing different hydrophobic compounds or nanoparticles in the organic droplet phase of the O/W emulsion. For example, a donor fluorophore and an acceptor fluorophore were both dissolved in chloroform. The chloroform solution was then emulsified into an aqueous phase containing PVA (80% hydrolysed, Mw = 10 K) and Brij 58 by sonication. Freeze‐drying of the emulsion produced dual‐component fluorescent nanoparticles (<200 nm) with varying compositions. These nanoparticles could be used for the investigation of cellular internalization by Caco‐2 cells and macrophages by fluorescence (Förster) resonance energy transfer (FRET). The breakdown of the dual‐component nanoparticles could lead to the loss of FRET signals. The extensive whole‐particle internalization was confirmed. A mechanism for enhanced in vivo pharmacokinetics was proposed through further study on the cellular permeability across Coco‐2 monolayers [111]. In another study, three‐component nanoparticles containing OR, a hydrophobic polymer, and oleic acid‐coated Fe3O4 nanoparticles (15–20 nm) were prepared by a similar approach. The three hydrophobic components were dissolved/dispersed in toluene, which was then emulsified by sonication. The superparamagnetism from iron oxide nanoparticles was retained. Ibuprofen was also incorporated into the nanoparticles and evaluated for controlled release [112].

Exciting progress has been made in the use of solid drug nanoparticles in terms of biological evaluations. Efavirenz, a non‐nucleoside reverse transcriptase inhibitor, is considered to be a first‐line global therapy for new cases of human immunodeficiency virus (HIV) infections. But this drug has low water solubility (<10 μg ml−1) and hence a large dose is required with increasing cost and side effects. The efavirenz nanoparticles were prepared by freeze‐drying of O/W emulsions (chloroform solution emulsified into aqueous solution containing various surfactants and polymers). The nanoparticles with sizes <400 nm were formed with non‐ionic surfactants α‐tocopherol poly(ethylene glycol) succinate (TPGS) or Tween‐80 with PVA (80% hydrolysed, Mw = 9500). These efavirenz nanoparticles showed reduced cytotoxicity and increased in vitro transport through model gut epithelium. An approximately fourfold higher pharmacokinetic exposure after oral administration was observed in the study with rodents [113]. Ritonavir (RTV) is a HIV protease inhibitor (PI) and usually used with other PIs (e.g. lopinavir (LPV)) as a booster to increase the pharmacokinetic profiles. The RTV nanoparticles prepared by the emulsion‐freeze‐drying method showed augmented inhibition of human CYP3A4 in cell‐free systems and human primary hepatocytes. In addition, higher permeability across intestinal cell membranes and lower cytotoxicity were observed [114]. Very recently, a 160‐component library high‐throughput screening process was developed to evaluate the polymer and surfactant combinations based on the emulsion‐freeze‐drying approach. The study employed chloroform‐in‐water emulsions and initially focused on LPV nanoparticles (with 10% drug loading to the excipients) and then the LPV‐RTV (70% drug loading, LPV:RTV = 4 : 1) nanoparticles. PVA was found to be the most successful polymer in producing nanoparticles of high drug/excipient ratio, followed by hydroxypropyl methylcellulose (HPMC). This approach could screen a large quantity of solid drug nanoparticles in a short period, thus enhancing the process of discovering and identifying optimal formulations, and speeding up the progression for clinical manufacturing [115].

8.7 Solvent Evaporation Within Porous‐Soluble Polymers

The main limitation of the emulsion‐freeze‐drying approach is with the selection and suitability of the organic solvent. Firstly, to allow the emulsions to be readily freeze‐dried in a commercial freeze‐dryer, the freezing points should not be too low (usually greater than −50 °C depending on the grade of the freeze‐dryer) and ideally the vapour pressure should be high. Secondly, the solvent should be immiscible with water so that O/W emulsions can be formed. This excludes the commonly used polar organic solvents such as acetone, ethanol, methanol, and THF. As for pharmaceutical products, the solvents should be in the category of Class 2 solvents (Table 8.2) and more preferably in the category of Class 3 solvents (Table 8.3). However, there are not many organic solvents available that comply with these requirements. For the drug nanoparticles prepared by emulsion‐freeze‐drying, the solvents that have been used include cyclohexane, O‐xylene, chloroform, and also toluene. However, this situation may be made worse by the fact that many poorly water‐soluble drugs are only dissolved in polar organic solvent, i.e. they have very low solubility in water‐immiscible solvents.

To address this issue, a solvent evaporation method within porous soluble polymers has been developed to prepare organic and drug nanoparticles [116, 117]. Solvent evaporation from thin film on substrates can form different types of nanostructures and nanoparticles [118, 119]. It has also been successfully applied to form drug nanoparticles [120]. However, the surface area on a substrate is low. It will require a very large surface to produce a quantity of drug nanoparticles. This problem can be solved by the use of emulsion‐freeze‐dried porous polymers as the scaffold to hold up the solution and then allow the solvent to be evaporated. The O/W emulsions can be formed by an organic solvent emulsified into an aqueous solution containing a surfactant and hydrophilic polymer. Freeze‐drying of the O/W emulsions generates a highly interconnected porous water‐soluble matrix [107]. The interconnected porosity allows the rapid uptake of organic solvent and oils. The soaked polymer matrix can be dissolved in water in seconds by shaking, which releases the oil droplets and produces an O/W emulsion on demand [121]. Different oils, including mineral oil, soy oil, and perfluorocarbon, have been used to form O/W emulsions. The size and number of the droplets in the O/W emulsions are tuneable simply by varying either the soaking time or the porosity of the porous polymers [121].

The procedures for the preparation of organic nanoparticles by solvent evaporation within porous polymers are straight forward, as shown in Figure 8.11. This method has been demonstrated with OR and poorly water‐soluble drugs such as curcumin, carbamazepine, griseofulvin, and paclitaxel [116, 117]. The soluble porous polymers are prepared by freeze‐drying aqueous polymer solution or O/W emulsions. The porous polymers are then soaked in organic solutions, which are formed by dissolving the organic compounds or poorly water‐soluble drugs in polar organic solvents (usually ethanol or acetone) or non‐polar/less‐polar solvent with low freezing points (e.g. dichloromethane). After soaking for a defined time, the soaked porous polymers are left in a vacuum oven (mostly room temperature) or simply in a fume cupboard to allow the solvent to evaporate. Drug nanoparticles are formed within the porous polymer during the solvent evaporation. Like the dry materials obtained by the emulsion‐freeze‐drying approach, the polymers containing drug nanoparticles can be readily dissolved in water to produce stable aqueous nanoparticle suspensions. The sizes of the nanoparticles vary, depending on the polymer and/or the surfactant used. For examples, OR and curcumin nanoparticles of about 50 nm, carbamazepine nanoparticles of 130 nm, paclitaxel nanoparticles of 500 nm, and griseofulvin nanoparticles of 430 nm, have been successfully prepared by the solvent evaporation method. In general, the combination of suitable polymer, surfactant, drug, and the solvent needs to be optimized to form drug nanoparticles with smaller sizes (e.g. <300 nm) and high nanoparticle yields.

Figure 8.11 Schematic representation of the formation of drug nanoparticles by solvent evaporation within porous polymers.

Source: Qian et al. 2011 [116]. Reprinted with permission from Royal Society of Chemistry.

8.8 Summary

Nanomedicine has been extensively investigated to enhance stability, improve therapeutic efficacy, and reduce side effects. This has been highly effective for poorly water‐soluble drugs, where the poor solubility, high dosage, and low bioavailability have been the main issues. A variety of techniques are developed to produce drug nanoparticles, with wet milling and high‐pressure homogenization being extensively used in research and pharmaceutical production. After the introduction to bioavailability and delivery of drug nanoparticles, this chapter focuses on the freeze‐drying of solutions/suspensions and particularly the emulsion‐freeze‐drying approach. The freeze‐drying methods can be applied to heat‐sensitive and soft hydrophobic compounds, either in solution or emulsion approaches. The emulsion‐freeze‐drying approach can generate a stable porous soluble matrix containing hydrophobic drug nanoparticles, which can be instantly dissolved in water to produce stable aqueous nanoparticle dispersions. Similar to other pharmaceutical formulations, excipients such as hydrophilic polymers and surfactants are required to form emulsions, act as lyoprotectant, stabilize the drug nanoparticles in solid state, and facilitate fast reconstitution and stable reconstituted solution/suspensions.

Design and selection of excipients are crucial in the freeze‐drying approaches. This depends on the target organs/tissues where different sizes of drug nanoparticles are required. For example, extremely small nanoparticles are required to pass the BBB while submicron particles may be sufficient to be passively absorbed into the tumour tissues via the EPR effect. In other cases, it is mainly about the permeation across biological membranes and also has a sufficiently long circulation time. The targets or criteria of selecting suitable excipients are: (i) to produce the nanoparticles of required sizes; (ii) to facilitate bioabsorption; (iii) to maintain long circulation; (iv) to direct to the target tissue; and (v) to allow controlled or responsive release. Other import issues to be considered are biodegradability and elimination from the body when the therapeutic roles are completed.

Drug nanoparticles should be sufficiently characterized by physicochemical techniques and also in vitro characterizations. However, it is highly likely that the characteristics of the drug nanoparticles in vitro and in vivo can be considerably different. Therefore, in vivo characterizations and evaluations are essential for the consideration in clinical uses. Some important properties need to be considered, that may depend on the specific techniques employed. These include: (i) crystallinity (which affects stability and dissolution); (ii) dissolution rate in vitro and in vivo; (iii) the size of nanoparticles in systemic circulation (which can be very different from the sizes determined in vitro, due to adsorption of various proteins and other components in blood); and (iv) pharmacokinetic profiles and interaction with cells during permeation and circulation to the target cells.


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