Chemical Systems - Textbook PDF

2.2 Chemical Systems metabolism in the liver. The length of the micron-sized needles, placed in the surface of a solid support like silicon, is designed to penetrate the subcutaneous layer, creating micronsized channels through the skin that allow the localized deposit of large molecular weight mo...

2.2 Chemical Systems metabolism in the liver. The length of the micron-sized needles, placed in the surface of a solid support like silicon, is designed to penetrate the subcutaneous layer, creating micronsized channels through the skin that allow the localized deposit of large molecular weight molecules, without causing bleeding or pain and without reaching nerve fibers and blood vessels in the dermis [16, 17]. 2.2 2.2.1 Chemical Systems Polymer-Based Nanocarriers Polymers are, by definition, substances whose molecular structures are composed of a large number of repeated units bonded together. Moreover, polymeric systems can be subdivided into two groups: (a) natural polymers, such as the polysaccharide chitosan, proteins, and peptides, and (b) synthetic polymers, such as cyclodextrins and poly(ethylenimine) [18]. Synthetic cationic polymers constitute the polymeric vehicles that are most often used in gene therapy. This is explained by their interaction with the negatively charged nucleic acids, originating polyplexes (designation for the complex formed by a polymer and the nucleic acid) capable of protecting the cargo and enabling its intracellular delivery [18, 19]. There are several natural and synthetic polymers used in gene delivery, such as chitosan, β-cyclodextrin, poly(L-lysine), poly(ethylenimine), dextran, and dendrimers [20, 21]. Additionally, these polymers can be engineered in order to improve their delivery efficiency, namely by adding cell-­ targeting ligands, like transferrin or epidermal growth factor (EGF), that will increase cellular internalization, or by adding shielding reagents, such as polyethylene glycol (PEG), that will increase the bloodstream circulation times, enhancing the amount of circulating delivery vectors and consequently gene-modifying tools that reach the target cells [22]. Some of these polymers are commercially available in ready-to-­ use polyfection (gene therapy transfer using a polymer) reagents. 29 2.2.2 Lipid-Based Systems Presently, there are three main lipid-based vehicles used for gene therapy: (a) liposomes, (b) high-density lipoprotein (HDL)-mimicking systems, and (c) microvesicular systems (which include exosomes). Therefore, the pharmacological effect of the nucleic acids carried in these systems is a function of the pharmacokinetic, biodistribution, and drug release characteristics of those carriers. Liposomes Liposomes are micro- or nanoparticles composed of one or more lipid bilayers, with an aqueous core. These particles can be formed through a self-assembly process after addition of an ethanolic solution of lipids to an aqueous solution of nucleic acids. These vehicles were introduced as drug delivery agents in the 1970s [23] and have been adapted for nucleic acid delivery; their efficiency as delivery tools is dependent on physicochemical characteristics, such as lipid composition, size, net charge, loading efficiency, and stability [18, 23, 24]. There are different types of liposomes, with different physicochemical characteristics and consequently more appropriate for different applications; some examples are (a) cationic liposomes and (b) “Smart”/Trojan horse liposomes. Cationic Liposomes Cationic liposomes have a net positive charge and thus spontaneously associate with negatively charged nucleic acids through electrostatic interactions. This results in the entrapment of the large nucleic acid molecules into smaller lipoplex (designation for the complexes made up of liposomes and nucleic acids) particles [18, 25]. Cationic liposomes successfully mediate nucleic acids delivery in vitro because they can be formulated so as to exhibit a net positive charge, which triggers their association with the negatively charged cell membranes. Additionally, they possess fusogenic properties, increasing the escape of the carried nucleic acid molecules trapped in the endosomes, upon internalization, into the cytosol (endosomal escape), thus signifi- 30 cantly decreasing their lysosomal degradation [18, 25, 26]. Since 1987, when Felgner and collaborators used cationic liposomes for the first time for gene therapy applications [27], many different cationic lipids have been synthesized and used for nucleic acid delivery purposes. Cationic lipids are composed of a positively charged polar head group, a hydrophobic region, and a linker connecting the polar and the non-polar group. These domains play an important role on the transfection ability and on the toxicity of the resulting lipoplexes. For example, the cationic head group, which will complex the negatively charged nucleic acid, is composed of either single or multiple groups of primary, secondary, tertiary, or quaternary amines or guanidine or imidazole groups. The liposomes made with multivalent lipids condense and protect the nucleic acids more efficiently than monovalent lipids [28, 29]. However, increasing the net positive charge may result in a lipid-nucleic acid interaction so strong that it reduces complex dissociation, consequently decreasing the efficiency of the nucleic acid release to the cytosol. Additionally, multivalent cationic lipids are more prone to form micelles, which compromise the liposomal/lipoplexes stability and also lead to higher toxicity of the resulting complexes. Cytotoxicity is also dependent on the stability and biodegradability of the lipid used to form the liposomes; thus lipids with strong linkages are normally more toxic [29, 30] because they are more difficult to be metabolized. One example of such lipids is the dioleoyloxypropyl (trimethylammonium) chloride (DOTMA), which has ether linkages and is more toxic than lipids with more labile ester linkages such as 1,2-dioleoyl-3 (trimethylammonium) propane (DOTAP) [18]. The main advantages of positively charged lipoplexes as compared to more complex systems, such as Smart/Trojan horse liposomes, are (i) the high efficiency in delivering nucleic acids in vitro; (ii) having no restrictions on the size of the nucleic acid to be delivered; (iii) their simplicity, given that these lipoplexes are easier to manufacture; (iv) the lower costs associated with these systems; and (v) some of them being commercially available [18]. Concerning the draw- 2 Non-viral Vectors for Gene Therapy backs, one of their major limitations is that, generally, they cannot be considered for in vivo applications through intravenous administration. This is explained by their cytotoxicity, instability, tendency to aggregate (which can lead to micro-­ emboli and tissue ischemia occurrences), and poor biodistribution (poor distribution through the organism, which will not enable the therapeutic compounds to reach their molecular and cellular targets) [25, 29–31]. This in vivo limitation extends to an inefficient in vivo transfection, also caused by the interaction of the positively charged lipoplexes with blood components, such as serum proteins, which decrease the availability of lipoplexes that reach and transfect the target cells. At the same time, this triggers lipoplex destabilization through modification of the lipoplex surface size and charge. These charge and size changes enhance lipoplex accumulation in the lungs, liver, and spleen, which is also a cause of the poor biodistribution of these particles. Opsonization (activation of the complement system, which activates phagocytosis and an inflammatory response) of positively charged lipoplexes with plasma proteins and lipoproteins, such as albumin and high-­ density lipoprotein (HDL), causes their rapid plasmatic clearance by the mononuclear phagocyte system (MPS) players, the phagocytic cells [18, 25, 30, 31]. Thus, lipoplexes are efficient in vitro nucleic acid delivery tools; however, they have important in vivo limitations, and consequently new lipid-based delivery systems have been developed to overcome the in vivo biodistribution limitations of the lipoplexes. A successful example of lipoplexes used as drug vehicles for clinical practice is the recently approved patisiran (Onpattro™), developed by Alnylam Pharmaceuticals [32]. This drug was approved for the treatment of hereditary transthyretin-­ mediated (hATTR) amyloidosis (hATTR amyloidosis) in adults with stage 1 or stage 2 polyneuropathy and is the first ever RNAi therapeutic approved in the EU. Onpattro acts by specifically downregulating the transthyretin (TTR) messenger RNA (via RNA interference with siRNA) and subsequently leading to a reduction in serum TTR protein levels and tissue TTR protein deposits, which are associated with the 2.2 Chemical Systems pathology. The siRNA molecules are complexed in lipoplexes and delivered intravenously to the liver, the primary place of TTR protein production. Patisiran significantly improves multiple clinical manifestations of these patients [32–34]. 31 allowing, therefore, the preferential delivery of the Trf-targeted nanocarriers to cancer cells [35– 38]. Stimulus-sensitive liposomes deliver their cargo after the influence of a specific stimulus. There are several stimuli able to destabilize liposomes and promote the release of their content. Smart/Trojan Horse Liposomes These stimuli can be endogenous factors such as The ideal lipid-based systems for nucleic acids pH and redox conditions, or exogenous factors and drug delivery would consist in (i) long-­ such as magnetic fields, ultrasound, and light. circulating liposomes with extended permanence Temperature can either be an endogenous or in the bloodstream and (ii) with the capability to exogenous stimulus. Thus, different controlled specifically target the cells/organs to be treated. release liposomal-based systems can be formuThis would (i) increase their ability to reach the lated in order to specifically respond and deliver target cells in therapeutic doses and (ii) decrease their content upon stimulation. the secondary effects caused by the delivery of Some stimuli allow the distinction between therapeutic agents to healthy cells. normal and pathological tissues; one example of Long-circulating liposomes are frequently these endogenous factors is the intratumoral pH called “Stealth” or “Trojan horse” liposomes, value that in solid tumors is significantly lower given their ability to evade the immune system and (approximately 6.5). Thus, pH-sensitive liporemain in the bloodstream for longer periods. The somes that are destabilized below pH 7.4 will most frequent strategy used to increase the perma- allow specific cargo delivery to cancer cells. nence of liposomes in the bloodstream is through Additionally, tumors are also characterized by a the modification of the liposomes’ surface by local temperature rise. Thus, thermosensitive introducing hydrophilic polymers, such as liposomes can be designed in order to maintain PEG. The incorporation of PEG in the particle sur- their cargo at the physiological temperature of face will sterically prevent the interaction and 37 °C and release their cargo at temperatures binding of blood components (like the comple- higher than 40–45 °C [35, 39]. ment system elements) to the liposome surface, Exogenous stimuli can be applied from the preventing its opsonization and removal from the outside in the local to be treated in order to trigbloodstream by the reticuloendothelial system. ger the release of the liposomal content. These Other polymers have also been used to prolong carriers have an on-off drug release because the blood circulation times such as poly(acrylamide), structure (lipid bilayer) is affected when stimupoly(vinylpyrrolidone), poly(acryloylmorpholine), lated and this allows achieving a temporal and poly(2-ethyl-2-­oxazoline), poly(2-methyloxazo- spatial controlled release of its cargo [35, 39]. line), phosphatidyl polyglycerols, polyvinyl alcohols, and others [18, 35, 36]. High-Density Lipoprotein (HDL)-Mimicking Smart liposomes are characterized by their Systems ability to deliver their cargo to a specific cell pop- High-density lipoproteins (HDL) are a group ulation or under the influence of a specific stimu- of lipoproteins including chylomicrons, very lus. Targeting nanocarriers to a specific cell low-density lipoproteins (VLDL), and the low-­ population is often carried out with the aid of density lipoproteins (LDL). Lipoproteins are specific ligands that are incorporated into the spherical particles composed of a hydrophobic nanocarrier surface. These ligands specifically core comprising triglycerides and cholesteryl recognize and interact with certain cell surface esters covered by a shell of apolipoproteins, components allowing the cargo to be selectively esterified cholesterol, and phospholipids. The delivered to those cells. One example of such different classes of lipoproteins differ in their ligands is the transferrin (Trf) protein, given that size, lipid and apolipoprotein composition, and in the Trf receptor is overexpressed in cancer cells, their specific functions. Generically, lipoproteins 32 are responsible for the transport of lipids in blood circulation, targeting different cell types through specific cellular receptors [40]. Endogenous HDL are nanoparticles with 6–13 nm in diameter, composed of apolipoproteins and lipids, with a density of 1.063–1.210 g/ mL that function has a transport system [41, 42] for hydrophobic molecules such as cholesterol. The most abundant apolipoprotein in HDL is apolipoprotein A-1 (Apo A1), representing 70% of the protein content, and is essentially responsible for supporting the size and shape of these lipoproteins. The major role of HDL is to transport cholesterol from the peripheral tissues to the liver for catabolism in the process of reverse cholesterol transportation. However, HDL are associated with other activities, namely the transportation of miRNA, carotenoids, vitamins, and hormones; they are also associated with antioxidative, antiinflammatory, anti-apoptotic, anti-atherogenic, and immunogenic activities. Additionally, HDL have been related to a positive influence on the regulation of glucose homeostasis [40–43]. As these particles are endogenous, they are biodegradable, do not trigger immune reactions, and are not captured and removed from circulation by the reticuloendothelial system [40], features that make them very promising delivery systems. SR-B1 is the natural receptor for HDL; this receptor has the highest expression in the liver, which is the main target organ of HDL. Therefore, many of the HDL-based systems developed so far have been formulated to target the liver. However, SR-B1 can also be found in adrenal gland, ovarian, and placental tissues [42]; additionally, it has been reported that this receptor is also highly expressed in some cancers, such as prostate, breast, colorectal, ovarian, and nasopharyngeal cancer, which is explained by the requirement of high cholesterol levels on the part of the highly proliferating cancer cells. Thus, HDL can also be formulated to deliver anti-cancer drugs [42]; however, in this particular case, the use of HDL to deliver toxic anti-cancer drugs may trigger substantial hepatotoxicity caused by the accumulation of the drug in the liver, mediated by the natural delivery of HDL to this organ. One way to overcome this problem is to modify the HDL in 2 Non-viral Vectors for Gene Therapy order to add additional targeting ligands mediating cell-specific cargo delivery to its surface, such as folic acid and targeting antibodies, like the CD20 antibody that targets B-cell lymphoma cells [42, 44]. To deliver the negatively charged nucleic acids with HDL it is necessary to introduce in the formulation cationic lipids, such as N, N-dimethyl-N, N-dioctadecylammonium bromide (DDAB), which will form complexes with the genetic material, allowing its incorporation in these particles. Thus, HDL can be artificially prepared in order to efficiently deliver nucleic acids [44–46] to a specific cell population evading the reticuloendothelial system and promoting no immune reaction. Microvesicles-Exosomes Exosomes are membrane-based vesicles with 40–100 nm that belong to the group of extracellular vesicles, which also include microvesicles and apoptotic bodies. These vesicles are produced by almost all types of cells, being released to the extracellular space with the purpose of acting as intercellular communication vectors promoting the transport of different molecules between different cells at big distances. The cargo content and the membrane composition of exosomes are dependent on the cells of origin and their current state, namely on their differentiation stage and stress. Thus, in addition to their potential use as transport vehicles, exosomes also hold the potential of being implemented in clinical practice as disease’s biomarkers. In fact, exosomes are enriched in particular proteins, RNAs, and lipids. Some of these are used as exosomal markers, enabling the confirmation of exosome preparation/isolation. These include the Alix, flotillin, TSG101, and CD63 proteins and raft lipids such as cholesterol, ceramide, and glycerophospholipids with long and saturated fatty acyl chains [47–49]. Other proteins have been used as biomarkers; for example, it is known that, in Parkinson’s disease, exosomes act as spreading vehicles of the disease, performing the transport of α-synuclein from diseased cells to healthy cells [50]. Thus, the presence of this protein in exosomes is an indication of the disease. 2.2 Chemical Systems Because exosomes are vesicles naturally produced by cells and their size allows a reasonable cargo transport between cells, exploring these vesicles as drug and nucleic acid delivery agents is logical. However, these vesicles still have several drawbacks, in particular the heterogeneity between batches and within the same sample, as different subspecies exist among exosomes isolated from the same batch of cells. The differences are related to size and composition, namely with respect to lipid, protein, and noncoding RNA content. As most of the identified noncoding RNAs in exosomes have unknown biological functions, and therefore the full impact of these molecules in the target cells is unknown, there is also a risk of triggering unpredictable secondary molecular effects. Additionally, in a systemic administration, exosomes have a poor pharmacokinetic profile, because they are captured in the liver, spleen, and lungs [47, 48]. 33 inorganic-based delivery systems are their easy functionalization, low cytotoxicity, and high biocompatibility [51]. Metal Nanoparticles Metal nanoparticles have been used as gene delivery vectors and noble metals such as gold and silver are some of the materials that are commonly used in their composition. These metals have characteristic surface plasmon resonance [52, 53] that allows specific sensoring; additionally, their surface is easy to be biofunctionalized and to be applied in specific applications such as light-based triggered events [51]. The major advantages of these systems are that they are easy to synthesize, they have a well-defined composition and good biocompatibility profile, and they are easy to track upon administration using techniques such as fluorescence resonance energy transfer (FRET) [54]. Quantum Dots Quantum dots (QD) are semiconductor crystals with sizes ranging from 1 to 20 nm [51, 54]; these Many inorganic compounds (Table 2.3) have systems are clusters of hundreds to thousands of been used to make inorganic nanoparticles, which atoms arranged in binary (e.g., CdSe, GaAs, have different physical and chemical characteris- InAs, SiC) or ternary compounds (e.g., InGaN, tics and capabilities depending on their composi- InGaP, InGaAs) [55]. QDs are used as fluorestion. The most often used inorganic materials are cence probes and have several advantages as magnetic compounds such as iron oxides, previ- compared to organic fluorophores, such as high ously described in the magnetofection section, brightness, longer fluorescence lifetime, and betmetal nanoparticles, quantum dots, graphene-­ ter photostability [51, 54]. These systems can be based systems such as carbon nanotubes, and engineered in order to become very efficient biosilica nanoparticles. The advantages of these sensing platforms with high specificity and 2.2.3 Inorganic Materials Table 2.3 Main advantages and disadvantages of inorganic materials used in gene therapy delivery Metal nanoparticles Quantum dots Carbon nanotubes Silica-based systems Advantages Easy to synthesize; well-defined composition; good biocompatibility; easy to track upon administration High specificity and sensitivity of tracking; can be functionalized to specific intracellular delivery of nucleic acids Small size; chemical inertness; high drug capacity; controlled drug release ability Good drug loading capacity; can be functionalized to have increase circulating times, targeting properties, and cellular uptake; good storage stability; low toxicity; unexpensive and easy preparation in large amounts Disadvantages Require specialized equipment Complex production Complex production; poor solubility in aqueous solutions, reducing the applications in biological systems Could lead to hemolysis and metabolic deregulation 34 s­ ensitivity used in bioimaging to detect specific targets such as nucleic acids [55]. Additionally, they can be designed for gene delivery purposes; the classical approach is the conjugation of the nucleic acids and a targeting ligand on the surface of the quantum dot core. This will combine in one system the fluorophore properties and the therapeutic functions (theranostics) [56], allowing, for example, the tracking of the intracellular delivery of the nucleic acids [54, 55] mediated by these systems. 2 Non-viral Vectors for Gene Therapy large quantity [53, 54, 58]. Nevertheless, probably the most attractive property of these systems is the ability to store and release a big variety of drugs and to provide a big surface to store the drugs and nucleic acids, allowing the incorporation of hydrophilic and hydrophobic molecules; the latter are particularly difficult to be delivered by other systems [53]. A classical surface modification that is made to silica nanoparticles is the introduction of cationic components (such as PEI, dendrimers, and cationic lipids), which will complex the negatively Carbon Nanotubes charged nucleic acids; the genetic material will, Carbon nanotubes are made up of one or more therefore, be adsorbed at the nanoparticle surface sheets of graphene with cylindrical shape. This and, consequently, will not be totally protected system can be formulated as (i) multiwalled car- from nucleases. This problem was overcome by bon nanotubes, exhibiting two or more cylindri- the design of silica nanoparticles with bigger cal graphene sheets centrically arranged and pores (>15 nm) whose surface was functionalized displaying 4–30 nm of diameter, or as (ii) single-­ in order to introduce positively charged primary walled carbon nanotubes, composed of a single amine groups, providing a large loading capacity graphene sheet, having 0.4–3 nm of diameter [51, and nuclease protection [59]. The loading capa54]. The small size, chemical inertness, high drug bility is a function of the roughness, pore size, and loading capacity and controlled drug release abil- the nature of the surface functionalization; accordity are some of the biggest advantages of this sys- ingly, thiol-­modified particles, followed by mixed tem. Additionally, it is also possible to attach thiol- and amino-functionalized silica nanopartibiomolecules and fluorescent probes to the car- cles, have the highest loading ability. bon nanotubes, improving their delivery properBesides the incorporation of targeting ligands, ties and enabling the study of the cellular delivery it is also possible to functionalize silica-based process [54, 57]. Moreover, carbon nanotubes systems in order to obtain nanoparticles respondcan form stable complexes with nucleic acids ing to pH, redox changes, and external stimuli, mediating their efficient delivery. However, the such as near-­infrared light, which can be used in poor solubility in aqueous solutions reduces the the development of less invasive diagnose methapplications in biological systems [51]. ods [58].The major disadvantages of silica-based nanoparticles are (i) the observed hemolysis Silica-Based Systems caused by its interaction with the surface of the Silicon dioxide (SiO2), or silica, nanoparticles phospholipids of the red blood cell membranes have been widely used to deliver nucleic acids, and (ii) the induction of metabolic changes resultdrugs, and dyes. These particles can be modified ing in melanoma [60–62]. in order to manipulate their size, shape, and porosity; it is also possible to functionalize their surface through the conjugation of several mole- This Chapter in a Nutshell cules such as targeting ligands and polymers that confer stealth properties. Thus, it is possible to • Gene-manipulating tools such as siRNA or modify them in order to obtain nanoparticles DNA have a very limited bioavailability, with long circulating times, favorable targeting because they are degraded by nucleases, do properties, good drug loading capacity, adenot efficiently cross cell membranes, and are quate cellular uptake profiles and low toxicity. eliminated from the bloodstream. Additionally, these systems have good storage • For the clinical implementation of gene therstability and are cheap and of easy preparation in apy, the development of vectors that efficiently