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Chapter 9
Inclusion Complexes in Drug Delivery and Drug Targeting: Formation, Characterization, and Biological Applications Rajesh K. K. Sanku,1 Ozlem O. Karakus,1,2 Monica Ilies,3 and Marc A. Ilies*,1 1Department
of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140, United States 2Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, 1 Discovery Drive, Rensselaer, New York 12144, United States 3Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, Pennsylvania 19104, United States *E-mail:
[email protected].
Host–guest complexation based on non-covalent interactions can be successfully exploited for drug and nucleic acid delivery purposes. The chapter covers the main classes of hosts used for these applications, namely cyclodextrins, calixarenes, and cucurbiturils and reveals the main representatives and their physicochemical characteristics that modulate the complexation process. It also highlights relevant drugs and nucleic acid complexes, together with the dysfunctions and diseases where the host–guest complexes have been successfully used.
1. Introduction Supramolecular chemistry was defined by Lehn as “the chemistry beyond the molecule,” in which chemists generate molecular assemblies through noncovalent bonds (1). Supramolecular structures are based on electrostatic forces [(ion–ion, dipole–dipole, H bonding (4–120 kJ/mol)], cation–π interactions (5–80 kJ/mol), dipole–dipole, van der Waals forces (< 5 kJ/mol), π–π interactions (0–50 kJ/mol), and hydrophobic and other non-covalent interactions (2–6). Particularly © 2019 American Chemical Society
interesting for pharmaceutical applications are the host–guest complexes, made by direct association between macrocyclic molecular hosts and various drugs or by pharmaceutically useful entities as guests, to yield supramolecular nanocarriers and nanomedicines, bioresponsive materials, new imaging systems, biosensors, for example (3, 7, 8). These host–guest complexes are based on the reversible association of a large, usually cavity-bearing, molecule (the “host”) with a smaller molecule that is encapsulated within the host cavity (the “guest”). The interaction involves a complementarity between the physicochemical parameters of the cavity of the host (e.g., size, shape, charge, hydrophobicity) and the corresponding parameters of the guest molecule in order to ensure a good fit and a thermodynamically stable complex. Other important factors that can modulate the formation and stability of the host−guest complexes are the environmental conditions such as the solvent, pH, and temperature, which are frequently used to provide stimuli-responsiveness of the host−guest systems (6). The host, essentially a container-shaped molecule or cavitand, can include naturally derived compounds such as cyclodextrins (CDs) and synthetic macrocyclic molecules such as calixarenes (CAs), pillararenes (PAs), and cucurbiturils (CBs) (Figure 1) (7). The size comparison of those macrocyclic molecules is given in Table 1 (7).
2. Thermodynamics of Host–Guest Complexation When combined together, the host and the guest can associate spontaneously, driven by the decrease in the Gibbs free energy (Figure 1a) (9). Besides mixing molecular solutions of the host and of the guest, the neutralization, co-precipitation, slurry mixing, kneading and grinding are other common methods that can be used for generating host–guest complexes (10). The complexation process and the stability of the host–guest complex are characterized by the complexation constant, Kc, which can be determined by a plethora of kinetic and thermodynamic measurement techniques. Thus, UV-visible light, fluorescence, and NMR spectroscopies as well as circular dichroism, calorimetric, and potentiometric methods are used for determining Kc and validating the stoichiometry of the components in the complex. Other methods include partitioning methods (octanol/water partitioning experiments or HPLC-based), stopped-flow kinetic measurements, ultrasonic relaxation methods, for example (8). The association of the two partners sometimes occurs with a rearrangement of their 3D structures or morphologies toward more stable states/conformations in complexed form due to the free energy contribution of van der Waals forces, hydrogen bonding, release of conformational strain, charge-transfer interactions or the release of high-energy water bound in the cavitand host (8, 11–14). The formation of the host–guest complex is reversible and independent of the chemical properties of the host or guest molecule (8).
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Figure 1. Host–guest complex formation: (a) schematic depiction of a cavitand (host) interaction with a smaller molecule with physicochemical properties matching its cavity properties (guest) to generate a stable host–guest complex characterized by the complexation constant (Kc); (b) schematic representation of different cavitand host species, showing the repeating structural unit and the overall shape of the host molecule.
Table 1. Molecular Parameters of Most Commonly Used CDs, CAs, PAs, and CBs Host molecule
Molecular parameters Internal diameter
External diameter
Height
(mm)
(mm)
(mm)
α-CD
0.57
1.37
0.78
α-CD
0.78
1.53
0.78
γ-CD
0.95
1.69
0.78
CA[4]
0.30
0.59
1.175
CA[6]
0.76
0.496
1.624
CA[8]
1.17
1.923
2.24
PA[5]
0.47
1.35
0.78
PA[6]
0.58
1.52
0.78
PA[7]
0.71
1.69
0.78
CB[5]
0.44
0.24
0.91 Continued on next page.
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Table 1. (Continued). Molecular Parameters of Most Commonly Used CDs, CAs, PAs, and CBs Host molecule
Molecular parameters Internal diameter
External diameter
Height
(mm)
(mm)
(mm)
CB[6]
0.58
0.39
0.91
CB[7]
0.73
0.54
0.91
CB[8]
0.88
0.69
0.91
3. Cyclodextrin-Based Host–Guest Complexes Of all host molecules used for generation of host–guest (inclusion) complexes, CDs are the most used because they are derived from natural sources and are usually highly soluble, commercially available in large amounts, relatively inexpensive, and possess a very low toxicity. Sometimes called cycloamyloses, CDs are cyclic sugars composed of α-(1→4)-linked d-glucopyranose units that can be produced at industrial scale by enzymatic degradation of starch using CD glucosyltransferases (CGTases). Each representative can be isolated from the reaction mixture in high purity through the use of specific precipitation agents such as n-octanol/decanol for the α-CD (6 glucose units), toluene for β-CD (7 glucose units), and cyclohexadecenol/cyclohexadec-8-en-1-one for γ-CDs (8 glucose units), respectively (3, 15). Higher oligomers can be separated only via chromatographic techniques, due to the absence of selective precipitation agents. Although larger CDs—containing up to 26 glucose units and beyond (16)— have been reported, the α-, β-, and γ-CDs and their derivatives are responsible for 95% of all applications, including pharmaceutical formulations, and therefore will be the only ones detailed in this chapter (Figure 2, Table 2) (3, 16–20).
3.1. Structure and Properties of CDs X-ray and neutron beam crystallography have revealed the structure of α-, β-, and γ-CDs as hollow truncated cones or tori (Figure 2) (16, 17). The primary carbon atoms from position 6 of the glucopyranose ring are located on the narrow (or primary) face of the truncated cone and can be derivatized relatively easily, whereas those located at the wider entrance (the secondary face) are secondary Catoms from positions 2 and 3 and less prone to undergo chemical transformations (Figure 2).
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Figure 2. (a) Main CD representatives, revealing (b )their toroidal structure and the orientation of the hydroxyl groups or α-d-glucopyranose units toward the exterior of the torus (hydrophilic, blue) and of the methinic hydrogens toward the interior of the torus (hydrophobic, red); the torus height (TH), outer diameter (OD) and internal diameter (ID) are presented for each representative in Table 2; (c) top and (d) side 3D view of β-CD, showing the hydration water inside and outside the cavity (constructed with RasMol for Windows, v. 2.7.1.1, based on Cambridge Crystallographic Data Center CSD Entry BCDEXD03 (17)).
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Table 2. Some Physicochemical and Biological Characteristics of CDs α-CD
Cyclodextrin
γ-CD
β-CD
Number of α-glucopyranose units
6
7
8
Molecular weight
972
1135
1297
Internal diameter (Å)
4.7–5.2
6.0–6.4
7.5–8.3
Outer diameter (Å)
15.3
16.6
17.2
Height of CD torus
7.8
7.8
7.8
Water solubility (g/L at 24 °C)
145
18.5
232
Cavity volume (Å3)
174
262
427
Cavity volume in 1 g CD (mL)
0.1
0.14
0.2
Water molecules in cavity
6
11
17
Moisture content (w/w)
10.2
13.0–15.0
8–18
Calculated clogP (octanol/water)
–7.8
–10.7
–12
Conc. (mg/mL) for 50% red blood cell hemolysis
11.7
7.8
32
The hydroxyl groups of the glucose units are oriented toward the outside surface of the truncated cone, making the compounds water soluble, while methinic protons of the glucose monomers are located inside the cavity, which is relatively hydrophobic (7). The secondary OH groups from the glucopyranose repeating units form an intramolecular H-bond network. This network is perfectly formed in β-CDs, which, as a consequence, have the lowest solubility in water among the three common types of CDs. α-CDs have one glucopyranose in a distorted position and in γ-CDs the glucopyranosyl units are not coplanar; both conditions perturb the intramolecular H-bond network, favoring H-bonding with water molecules and thus dramatically enhancing the water solubility of these representatives (Table 2) (18). The size of the primary and secondary sides of the CDs depends on the number of glucose repeat units, whereas the depth of the hollow cavity is 7.8 Å for all the three CDs, given by the height of the truncated cone. The inner diameter of the unmodified CDs ranges from 4.7 to 8.3 Å for α- to γ-CDs (Table 2). Thus, a large variety of inclusion complexes can be formed with guests having molecular sizes within this range. The guests range from proteins and other polymer chains to cationic or anionic guests and small neutral molecules. Upon formation of the inclusion complexes, some properties of the guest molecules, such as solubility and reactivity will change and this phenomenon constitutes the basis of many applications involving CDs-based complexation. A dynamic equilibrium occurs in solution between the host, the guest, and their complex (Figure 1). However, many drugs are slightly longer than the cavity of the common CDs. Moreover, α- and β-CDs display some toxicity because they are reabsorbed and concentrated in the renal tubule. Because CDs can also complex and 192
extract cholesterol and lipids from cells, the process of continuous reabsorption and concentration at the level of kidneys changes the natural distribution of amphiphiles at the level of these organs and generate mild toxicity upon long-term exposure (21). The LD50 values for α-CD, β-CD, and γ-CD in rodents (i.v. administration) are approximately 1.0, 0.79, and > 4.0 g/kg, respectively. Parenteral administration of β-CD leads to precipitation of its aggregates in the kidney, resulting in renal tubule damage (22). Functionalized β-CDs can mitigate these problems. To enhance drug encapsulation and release ability, and to further improve water solubility and lower the toxicity, CDs were chemically modified. One such chemically modified CD is 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a hydroxyalkyl derivative provided as an alternative to α-, β-, and γ-CDs, with improved water solubility properties and reduced toxicity (23, 24). It was introduced over 35 years ago by Janssen and the NIH as a more water-soluble alternative to β-CDs and quickly became an approved solubilizer and stabilizer for oral and parental drug formulations in the U.S. and European Pharmacopoeias after Marcus Brewster’s pioneering work toward the characterization of its properties and safety profile (24–26). It is synthesized by reacting β-CD with propylene oxide in alkaline conditions, controlling the degree of substitution (usually around 6/CD molecule) via reagent ratios and the amount of base used. The hydroxypropyl groups also prevent aggregation of the HP-β-CD in solution, a phenomenon frequently observed for β-CDs (24, 26). Another CD derivative with pharmaceutical applications is the sulfobutylether β-cyclodextrin (SBE-β-CD). The commercially used derivative contains an average of 6.6 sulfobutylether units per unit of β-CD and is administered as a sodium salt. Due to their specific physicochemical parameters and increased water solubility, both HP-β-CD and SBE-β-CD display reduced reabsorption at the level of the kidneys and, consequently, a lower toxicity as compared with the parent β-CD (21, 27). Other derivatives include the randomly methylated β-CD (RM-β-CD) and hydroxypropyl-γ-CD (HP-γ-CD) (8, 18). Due to their good water solubility and low toxicity, these CDs are categorized by the U.S. Food and Drug Administration (FDA) as “generally recognized as safe” (GRAS) (6). Many other CD derivatives exist and are used in chemical and biomedical research and the field is continuing to expand. However, the GRAS status is not valid for all representatives given that chemical modification can dramatically change the physicochemical properties of the CDs (19, 22, 28). These chemically modified CDs need to be reassessed in terms of toxicity and their GRAS status needs to be confirmed. Representative examples are presented in the following sections. 3.2. CDs in Drug Formulations and Drug Delivery Systems The complexation of guest molecules by CDs in aqueous solution results in a substantial rearrangement of solvation water and the removal of the water molecules from the CD cavity and solvation water of guest molecules, with significant entropic gain. For relatively small hosts, a 1:1 complex is formed (molecular encapsulation, exemplified by a benzaldehyde-α-CD complex in Figure 3). Lipophilic molecules displaying a strong hydrophobic effect are preferred and the result is a substantial increase in the water solubility of these 193
compounds, which constitutes the basis for using CDs as solubility enhancers for lipophilic drugs. The nanometric dimensions of CDs ensure a high dispersion of lipophilic drugs in water, preventing their crystallization in aqueous media and enhancing their bioavailability. The (lipophilic) encapsulated drug can be released upon contact with the lipophilic cell membrane, with CDs acting as drug nanocarriers and delivery systems. Examples of CDs in drug formulations and delivery are presented in Table 3 (8, 19, 22).
Figure 3. The α-CD-benzaldehyde inclusion complex: (a) top and (b) side 3D view revealing the relative position of the guest molecule inside the cyclodextrin cavity and the hydration shell of the complex; notice the absence of hydration water inside the CD cavity (constructed with RasMol for Windows, v. 2.7.1.1, based on Cambridge Crystallographic Data Center CSD Entry BAJJAX (29)).
The range of host molecules is not limited to solid compounds. Liquid and gaseous compounds can be encapsulated in CDs and delivered in aqueous solutions. CDs can significantly reduce the volatility of low-boiling point liquids through host–guest complex formation, and thus can either mask unpleasant odors generated by them or encapsulate and slowly release pleasant odors/scents (30, 31). Water plays an important role in the formation and stability of the host–guest complexes of CDs. The affinity of hosts for their guests usually decreases when the solvent (water) is removed and decomplexation can occur, especially for compounds with low hydrophobicity, resulting in molecular dispersions of host and guests. The phenomenon is reversible and complexes are formed spontaneously when water is added to these dispersions. The very lipophilic drugs do not decomplex from their CD hosts even in dry form and the CD complex can, for example, be tableted, mixed, or milled as a single entity. This is particularly important for oils and lipophilic liquid drugs that can be converted into powders for tableting and for other solid formulations. 194
Table 3. Some Marketed Products Containing CDs. Source: Adapted with permission from ref (8). Copyright 2007 Elsevier. Cyclodextrin/drug
Trade name
Company
Formulation
α-CD Prostaglandin-E1
Caverject Dual
Solution (i.m./i.v. use)
Pfizer
Alprostadil
Prostavastin, Rigidur
Solution (i.v./i.a. use)
Ono, Schwarz, Ferring
Cefotiamhexetil·HCl
Pansporin T
Tablet
Takeda
Limaprost
Opalmon
Tablet
Ono
Benexate·HCl
Ulgut, Lonmiel
Capsule
Teikoku, Shionogi
Cephalosporin
Meiact
Tablet
Meiji Seika
Cetirzine
Cetrizin
Chewable tablet
Losan Pharma
Chlordiazepoxide
Transillium
Tablet
Gador
Dexamethasone
Glymesason
Ointment, tablet
Fujinaga
Dextromethorphan
Rynathisol
Synthelabo
β-CD
Diphenhydramine and chlortheophylline
Chewable tablet
Stada
Ethinylestradiol and drospirenone
Tablet
Yaz, Bayer
Iodine
Mena-Gargle
Solution
Kyushin
Meloxicam
Mobitil
Tablet, suppository
Medical Union
Nicotine
Nicorette
Sublingual tablet
Pfizer
Nimesulide
Nimedex
Tablets
Novartis
Nitroglycerin
Nitropen
Sublingual tablet
Nihon Kayaku
Omeprazole
Omebeta
Tablet
Betafarm
Prostaglandin-E2
Prostarmon E
Sublingual tablet
Ono
Piroxicam
Brexin, Flogene, Cicladon
Tablet, suppository
Chiesi, Aché
Tiaprofenic acid
Surgamyl
Tablet
Roussel-Maestrelli
Cisapride
Propulsid
Suppository
Janssen
Hydrocortisone
Dexocort
Solution
Actavis
Indometacin
Indocid
Eye drop solution
Chauvin
HP-β-CD
Continued on next page.
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Table 3. (Continued). Some Marketed Products Containing CDs Cyclodextrin/drug
Trade name
Formulation
Company
Itraconazole
Sporanox
Solution (oral and i.v.)
Janssen
Mitomycin
MitoExtra, Mitozytrex
Solution (i.v. use)
Novartis
Aripiprazole
Abilify
Solution (i.v. use)
BMS, Otsuka
Maropitant
Cerenia
Parenteral solution
Pfizer
Voriconazole
Vfend
Solution (i.v. use)
Pfizer
Posaconazole
Noxafil
Solution (i.v. use)
Merck
Ziprasidone mesylate
Geodon, Zeldox
Solution (i.m. use)
Pfizer
Carfilzomib
Kyprolis
Solution (i.v. use)
Amgen
Amiodarone
Nexterone
Solution (i.v. use)
Baxter
17β-Estradiol
Aerodiol
Nasal spray
Servier
Chloramphenicol
Clorocil
Eye drop solution
Oftalder
CardioTec
Solution (i.v. use)
Squibb Diagnost
Diclofenac Na+ salt
Voltaren Ophtha
Eye drop solution
Novartis
Tc-99 Teboroximea
CardioTec
Solution (i.v. use)
Bracco
SBE-β-CD
RM-β-CD
γ-CD Tc-99 Teboroximea
HP-γ-CD
The high affinity of lipophilic molecules to CDs can be further exploited to provide 3D-non-covalent interactions, when both the host and the guest are covalently linked to other chemical entities (e.g., small molecule, oligomers, polymers). Adamantane/β-CD interaction is a typical example: adamantane has a very high affinity for β-CD (Kc as high as 105 M−1, depending on substitution) (18), due to its high hydrophobicity and complementary size for the cavity of this CD (32). This strong host–guest association can be exploited for attaching drugs and targeting moieties (vide infra). Lipophilic azobenzene and ferrocene also display strong interactions with CDs. Their host–guest complexes are responsive to light and redox agents and constitute the base of several stimuli-responsive CD-based delivery systems (33–35). Besides the straightforward 1:1 complexation, one can observe 1:2, 2:1, 2:2, or even more complex associations, depending on the type of the CD used and on the size and molecular shape of the guest(s) (18, 22). 196
Based on all the above properties, it is obvious that CDs can act as efficient drug delivery systems (DDSs) given that they can complex a large variety of hydrophobic drugs, enhancing the drug’s water solubility, bioavailability, and stability (3). This is particularly important because prospective analysis has revealed that about 40% of the currently marketed drugs have a low water solubility and as much as 90% of drugs under current development can also be characterized as poorly soluble (19). Importantly, host–guest complexes of CDs with different drugs usually cause a significantly reduced irritation at the level of different tissues (e.g., gastrointestinal, ocular) based on the administration route as compared with the corresponding free drugs administered alone. It is known that DDSs can change the pharmacokinetics of drugs and can focus their activity on specific target tissues; this is valid for all CD-based DDSs with parental administration. DDSs can control the loading and release of drugs and can also isolate and protect them from inactivation en route to the target tissue or organ where they can elicit the desired therapeutic effect. Through host–guest complex formation, CDs can significantly reduce the free drug concentration in solution, which is particularly important for drugs that have unpleasant smells and/or bitter taste. Importantly, through the same mechanism, CDs can reduce or completely prevent drug–drug and drug–additive interactions (10, 19, 22).
3.3. CD-Based Polymeric DDS CDs can be attached to a polymer either to enhance the water solubility of the polymer backbone and/or to transform the polymer into a drug delivery system via pendant CDs (3). Many strategies are known for grafting CDs onto polymeric backbones, such as polymerization of monomers containing CD or CD cross-linking by alkylating agents (22). The complexation, transport, and delivery of the drug can be done either directly, or via a strongly complexing host such as adamantane on which drug molecules are covalently attached. In this context, Maciollek et al. (36) synthesized a 5-fluorocytosine conjugate bearing an adamantyl moiety and introduced its non-covalent inclusion complex with a CD-decorated poly(N-isopropylacrylamide) polymer as a DDS model. Their complex formation was validated by UV-visible light and turbidity analysis. The release of the drug under treatment with α-amylases proved to be relatively fast, with about 50% of the drug being released in 72 h. Because fluoropyrimidines and especially 5-fluorouracil (5-FU) represent frequently used chemotherapeutic agents, with proved tumor-inhibitory properties, this technology represents a promising achievement and a model for how CD-based host–guest DDSs can be used toward cancer treatment (36). CD-containisng polymers were also used successfully in nucleic acid delivery. Thus, in a classic example, Davis’s group synthesized β-CD-containing cationic copolymers for pDNA delivery by copolymerizing difunctionalized β-CD monomers (AA) with different difunctionalized comonomers (BB) in order to generate a CD-containing AABBAABB copolymer (37). Several structure–activity correlation studies revealed the importance of proper location of positive charges relative to the β-CD cup and their optimum spatial location on 197
the copolymer backbone (37, 38). The most efficient β-CD-containing cationic copolymer was found to be β-CD-P6 (Mn ~ 5.1 KDa), which displayed also a good cytotoxic profile (Figure 4). Importantly, the toxicity of the new β-CD-containing cationic copolymers was found to be three orders of magnitude lower than the toxicity displayed by their congeners in which bis(aminoethylthioβ-CD) difunctional monomer was replaced with a hexamethylenediamine one (38). The same team developed a method for modifying the pDNA polyplexes generated from linear CD-containing copolymers such as β-CD-P6 to make them more suitable for in vivo use. Thus, polyplexes were further PEGylated on their surface via host–guest interaction of β-CDs of the polyplex with PEGylated adamantane conjugates (AdCH2NHCOPEG5000), yielding “stealth” polyplexes that displayed reduced interactions with serum proteins and were thus amenable for in vivo pDNA delivery (Figure 4) (39).
Figure 4. Structure of a β-CD-P6 copolymer and of adamantane derivatives used for PEGylation of its corresponding lipoplexes via host–guest complexation in order to generate stealth properties and for targeting to asialoglycoprotein from the surface of liver hepatocytes (22, 37–39).
The same strategy was also used to target polyplexes toward hepatocytes using a modified PEG-adamantanepeptidyl conjugate (Ad-CONH-EAEAEAEAC(SCH2CH2SO2PEG3400CONH-Gal)-COOH. The targeting moiety contained the adamantane moiety for attaching the conjugate on CD-displaying polyplexes, an anionic peptide for generating a negative surface charge for polyplexes—in order to reduce interaction with negatively charged proteins in vivo—PEG5000 to sterically stabilize the polyplexes and further reduce nonspecific protein absorption in vivo, and galactose as a ligand targeting asialoglycoprotein receptors (ASGP-R) on the surface of hepatocytes (Figure 4). Biological testing on the Hep-G2 cell line in the absence or the presence of asialofetuin (a natural ligand for the ASGP-R) confirmed the receptor-mediated endocytosis of the targeted polyplexes in a selective manner, thus validating the novel targeting design based on host–guest interactions (39). In addition, transferrin-PEG-adamantane conjugates were synthesized via several synthetic 198
strategies and were used for targeting similar CD-containing polyplexes to tumors expressing the transferrin receptor on their surface (Figure 4) (40). Following the success of DNA delivery, Davis’s group tested their CD/adamantane host–guest-based targeted system for siRNA delivery, using an siRNA targeting the EWS/Fli1 fusion oncogene in a murine model of metastatic Ewing’s sarcoma (41). The good efficiency demonstrated by the targeted polyplexes constituted the foundation for the creation of a company, Calando Pharmaceuticals, which was charged with the translation of the new delivery platform, denominated RONDEL (from RNAi/Oligonucleotide Nanoparticle Delivery), toward clinical evaluation in human cancer patients. Research efforts led to the CALAA-01 siRNA delivery system targeting the M2 subunit of ribonucleotide reductase (RRM2) (42). The ribonucleotide reductase (RR) is a key therapeutic target for DNA replication-dependent diseases, such as cancer, implicated in cell division. The authors identified the optimal anti-RRM2 siRNA sequence (siR2B+5) via computational methods, and additional “tiling” duplexes, with significant knockdown of RRM2 protein in cultured cells and a pronounced antiproliferative activity in various cancer cells of human origin and from other species (i.e., mouse, rat, monkey) (42). The translation of the nanosystem in vivo, done in cynomolgus monkeys at doses of 3 and 9 mg siRNA/kg, showed that the nanoparticles were generally well tolerated, with no clinical signs of toxicity clearly attributable to treatment (43). Elevated levels of blood urea nitrogen and creatinine were observed at a high dose of 27 mg siRNA/kg, together with mild elevations of alanine amino transferase and aspartate transaminase, indicating some kidney and liver side effects at this high dose. Moreover, at 27 mg siRNA/kg, increased IL-6 levels were observed in all animals and were accompanied by an increased IFN-γ (noticed in only one animal), indicating a mild immune response and defining the upper level of the therapeutic window for this technology. Importantly, the study revealed that multiple administrations over 17–18 days were possible without significant antibody generation against the human transferrin component of the formulation or hypersensitivity reaction, thus confirming an excellent safety profile for the nanoformulation to nonhuman primates (43). Subsequently, Calandro issued an investigational new drug (IND) application to the FDA and received approval to initiate a phase I trial of CALAA-01 in patients with solid tumors in 2008 (44). In 2010, interim human clinical data from this study was published (45), providing evidence of the efficient induction of RNA interference in humans from a systemically delivered siRNA via RONDEL technology. Thus, tumor biopsies from melanoma patients obtained after treatment revealed a dose-dependent localization of the nanoparticles intracellularly, together with a clear reduction in the RRM2 mRNA and protein levels as compared with the same tissue prior to nanoparticle administration (45). 3.4. Other CD-Based Nano-Carriers A plethora of modified CDs have been synthesized in recent decades and used for drug and nucleic acid delivery, with some of them detailed in a recent review (28). Of considerable interest are the amphiphilic CDs, which can self-assemble 199
in different supramolecular assemblies (e.g., vesicles, rods, spherical micelles), depending on the packing parameter. For some representatives, biomedical applications are foreseen for the nanoencapsulation of drug molecules, either in the hydrophobic interchain volumes and, more traditionally, in the nanocavities of the amphiphilic CDs, thus serving as drug carriers or pharmaceutical excipients in anticancer phototherapy and gene delivery, for example (Figure 5) (28).
Figure 5. Schematic presentations of nanoarchitectures and nanoparticles involving amphiphilic cyclodextrins (28).
4. Calix[n]arene-Based Host–Guest Complexes Within the family of complexing macrocyclic molecules, calix[n]arenes have established their own place as another major class of macrocyclic hosts, with extensive and comprehensive applications of their host–guest complexes. The cup-like shape of their cavity (which also give their name) and the conformational flexibility of these molecules enable them to act as efficient host molecules for various molecules with biological or pharmaceutical utility. A size comparison between various CDs and calix[n]arenes is presented in Table 1. 4.1. Structure and Properties of CAs The calix[n]arenes are basket-shaped macrocyclic compounds synthesized by the condensation of p-alkyl-substituted phenols and formaldehyde in basic media. Because CAs with even numbers (n = 4, 6, and 8) can be synthesized in a one-step reaction and can be easily purified, they have been widely investigated. Although CAs are reported with 3–20 repeating phenolic units, the most frequently used are 200
the tetramers CA[4] and hexamers CA[6]. The most common conformation of CAs is a truncated cone shape with all the phenoxy groups positioned at the lower rim. This conformation is preferred by CAs due to the intramolecular hydrogen bonding among the phenolic groups (46). The hollow cavity of CAs is delimited by the two rims, at the primary and secondary sides. Various conformations of calix[n]arenes can be locked in place via substitution of the two rims with bulky groups (e.g., t-Bu-, HOSO2-, placed in the para position of phenolic units, or via replacement of –OH groups with bulkier groups), which increases the rotational barrier (47, 48). Cavity complexation is the main feature of macrocyclic hosts and generates functional supramolecular architectures in a manner similar to CDs. Compared with other common macrocycles such as CDs and CBs, for example, underivatized CAs are not as suitable for the generation of inclusion complexes due to their very low water solubility. This low solubility has negatively impacted their potential biological applications for a long time (49). Host–guest applications of CAs use representatives derivatized with hydrophilic groups, which have a significantly higher water solubility as compared with parent molecules.
Figure 6. The 8-aminoquinoline inclusion complex of SCA[4] in side 3D-view revealing the relative position of the guest molecule inside the sulfonated calix[4]arene host and the conformation adopted by the host molecule (constructed with RasMol for Windows, v. 2.7.1.1, based on Cambridge Crystallographic Data Center CSD Entry JITGUP (60)).
Chemical modification of CAs can lead to both endo- and exo-complexation (49, 50). For instance, sulfonated calix[n]arenes have the ability to bind guests in their cavities in aqueous media (51, 52). Sulfonation of the CAs at the rim is another strategy explored to increase their solubility (53). Alternatively, functionalization with carboxylic acid groups onto the lower rim and the 201
functionalization of polar groups at the edge of CAs have been carried out for the same purpose (54). Through the use of click chemistry, a large number of cationic, anionic, and nonionic CA derivatives with good water solubility were recently synthesized and characterized (55–58). Sulfonated calix[n]arenes (SCAs) are characterized by an inner hydrophobic core and an outer amphiphilic surface induced by their benzene rings and sulfonic acid groups, respectively (Figure 6)—an advantage for designing delivery systems (59). Their ability to complex with hydrophobic guest molecules is doubled by their biocompatibility in vivo and in vitro.
4.2. Toxicity of the SCAs Toxicity constitutes a key parameter to consider when evaluating potential biological applications of host molecules. Several toxicology studies for SCAs are available, performed both in vivo and in vitro. Thus, Silva et al. showed that, p-sulfonato-calix[4]arene has zero haemolytic toxicity up to a 5-mM concentration (61). Coleman and co-workers studied the biodistribution and pharmacokinetics of p-sulfonatocalix[4]arene in mice and reported no acute toxicity. They also proved that p-sulfonato-calix[4]arenes do not pass through the blood–brain barrier. Almost no uptake of p-sulfonato-calix[4]arenes was observed in liver and spleen, which confers a solid base for the use of these hosts in biological applications (62).
4.3. Guest Binding Ability and Thermodynamics of SCAs Host–Guest Complexation Isothermal titration calorimetry (ITC) is a powerful method for investigating the association constants and the thermodynamic parameters of the inclusion complexes because it allows for monitoring the enthalpy and entropy changes during the complexation process. Studies of SCAs with different guest molecules (i.e., anionic, cationic, and neutral molecules) showed that the electrostatic and hydrogen-bonding interactions between host and guest molecules affect enthalpy changes positively (63). In general, hydrogen bonding, π-stacking, van der Waals and C–H–π interactions between SCAs and guest molecules mainly contribute to the enthalpy changes, whereas the conformation changes and desolvation effects contribute to the entropy changes during the host–guest complexation process (64, 65). Shinkai and collaborators calculated the association constants for aqueous complexes with SCAs by the NMR method. They found that sulfonatocalix[4]arenes and sulfonatocalix[6]arenes form 1:1 stochiometric host–guest complexes with trimethylanilinium chloride and adamantyltrimethylammonium chloride, whereas sulfonatocalix[8]arene forms 1:2 complexes (66). Comprehensive complexation studies of p-sulfonatocalix[n]arenes with amino acids and proteins have been performed by NMR spectroscopy and/or by the microcalorimetric titration technique (67). These studies demonstrated that p-sulfonatocalix[n]arenes can form complexes with α-amino acids, with 202
the aromatic or aliphatic side chain of the amino acid being localized in the CA cavity (68, 69). Silva et al. studied the binding of p-sulfonatocalix[n]arene derivatives to bovine serum albumin (BSA) using electrospray ionization (ESI)-mass spectrometry. Their team reported that the strength of the sulfonatocalix[n]arene-BSA interaction decreases with the enlargement of the sulfonatocalix[n]arene cavity size (70).
4.4. SCAs as Drug Delivery Systems Recently, these negatively charged molecules with diverse sizes and substitution patterns have been used to increase the solubility of many drug molecules (71). Thus, Yang et al. have studied the solubilization with p-sulfonatocalix[n]arenes of three low-solubility drugs, namely nifedipine, furosemide, and niclosamide, in aqueous solution (72). The authors have demonstrated that the molecular size of calix[n]arene influences the aqueous solubility of these drugs remarkably (72). Liu et al. studied the binding behavior of p-sulfonatocalix[4]arene with pyridinim guest ions by 1H-NMR spectroscopy and microcalorimetry (73). They found that the pyridinium molecule can bind into the calix[4]arene cavity with the N atom located close to hydrophilic sulfonic groups (73). Host–guest complexation properties of viologens (toxic bispyridinium derivatives) with p-sulfonatocalix[4]arenes, sulfonatocalix[5]arenes, and thiacalix[4]arenes have also been investigated by Wang et al. (74) They reported that viologen molecules could form stable inclusion complexes with all CA host molecules (74). Some other drugs used in host−guest complexation for delivery applications with SCAs are listed in Table 4.
5. Cucurbit[n]uril-Based Host–Guest Complexes 5.1. Structure and Properties of Cucurbit[n]urils Cucurbit[n]urils, abbreviated as CB[n], are self-assembled macrocycles synthesized by acidic condensation of n structural units of tetrahydroimidazo[4,5d]imidazole-2,5(1H,3H)-dione (glycoluril) and formaldehyde as a provider of methylene (-CH2-) linkers (88–90). The different number of glycoluril residues (n = 5–8, 10) determine a pumpkin-shaped macromolecular cavity of the same height, but with different diameters (Figure 7). The cucurbituril name of these cyclic compounds comes from cucurbita (Latin for gourd) (90).
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Table 4. Drugs Used for Complexation with SCAs, their Corresponding Hosts, and the Biological Applications of the Host–Guest Complexes Drug
Carriers
Biological application
Reference
Dinuclear platinum complexes
p-Sulfonatocalix[4]arene
Ovarian cancer treatment
(75, 76)
3-Phenyl-1H[1]benzofuro[3,2c]pyrazole
p-Sulfonatocalix[6]arene and p-sulfonatocalix[8]arene
Tyrosine kinase III inhibitor
(77)
Carvediol
p-Sulfonatocalix[6]arene and p-sulfonatocalix[8]arene
Management of hypertension
(78)
Doxorubicin
p-Sulfonatocalix[6]arene
Antibiotic
(79)
Paclitaxel
Tetra-hexyloxy-psulfonatocalix[4]arene
Ovarian, breast, lung and colon cancer treatment
(80)
Tramadol·HCl
p-Sulfonatocalix[n]arenes
Analgesic
(81)
Nedaplatin
p-Sulfonatocalix[4]arene
Antineoplastic
(82)
Irinotecan·HCl
Tetra-hexyloxy-psulfonatocalix[4]arene
Colon cancer treatment
(83)
Mitoxantrone·HCl
Tetra-hexyloxy-psulfonatocalix[4]arene
Metastatic breast cancer and acute myeloid leukemia treatment
(83)
Procaine
p-Sulfonatocalix[6]arene
Local anesthetic
(84)
Fluconazole
p-Sulfonatocalix[n]arenes
Antifungal
(85)
Norfloxacin and Ciprofloxacin
p-Sulfonatocalix[8]arene
Antimicrobials
(51, 86)
Nifedipine
p-Sulfonatocalix[n]arenes
Calcium channel blocker
(72)
Topotecan
p-Sulfonatocalix[4]arene
Chemotherapeutic agent
(87)
Benzocaine
p-Sulfonatocalix[n]arenes
Local anesthetic
(47)
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Figure 7. The CB[6]-p-xylenediammonium chloride inclusion complex: (a) top and (b) side 3D- view revealing the relative position of the guest molecule inside the cucurbituril cavity (constructed with RasMol for Windows, v. 2.7.1.1, based on Cambridge Crystallographic Data Center CSD Entry CISWOQ (89)).
The discovery of the curcubituril family started in 1905 when Behrend and co-workers (88) first synthesized CB[6]. However, the X-ray crystal structure of the hexamer was elucidated by Freeman, Mock, and Shih only in 1981 (90). Kim and co-workers isolated CB[5], CB[7], and CB[8] in 2000 (91). Shortly after, CB[10] was reported (92) as a gyroscane-type CB[10]·CB[5]·Cl– 1:1:1 inclusion complex—an unusual “pumpkin-in-pumpkin” (or Russian Matrioshka doll) supramolecular assembly in which CB[5] and CB[10] are concentric, but not coaxial, while CB[5] moves freely within the spacious cavity of CB[10]. Interestingly, CB[5] also gets in or out of CB[10] freely, suggesting the possibility of CB[5] removal by competitive guest complexation. Indeed, a couple of years later, Isaacs’ group isolated CB[10] by using melamine diamine as a competitive guest to displace CB[5] (93, 94). Although CB[n]s with n = 13–15 have been reported more recently, CB[10]’s cavity remains the largest in the family, given that the too-large macrocycles adopt inevitably twisted conformations (Table 5) (94–97).
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Table 5. Physicochemical Parameters of CB[n]s. Source: Adapted with permission from ref (95). Copyright 2018 Elsevier. Parameter
CB[5]
CB[6]
CB [7]
CB[8]
CB[10]
Portal diameter, Å
2.4
3.9
5.4
6.9
9.5–10.6
Cavity diameter, Å
4.4
5.8
7.3
8.8
11.3–12.4
Outer diameter, Å
13.1
14.4
16.0
17.5
20.0
Height, Å
9.1
9.1
9.1
9.1
9.1
Cavity volume, Å3
82
164
279
479
870
Water solubility, mmol/L
20–30
0.018
20
0.01
