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Nanomedicines

Design, Delivery and Detection

The Royal Society of Chemistry

Design Considerations for Properties of Nanocarriers on Disposition and Efficiency of Drug and Gene Delivery

JOSE MANUEL AGEITOS, JO-ANN CHUAH AND KEIJI NUMATA

Enzyme Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan

1.1 Introduction

Delivery of drugs or genetic material into cells is one of the emerging areas of biotechnology. Nanoparticle (NP)-based drug carriers are especially interesting, due to their ability to deliver drugs inside target cells, thereby reducing the side-effects of non-specific treatments. Among the various cargoes that are transportable by NPs, genetic material allows the re-programming of cells both temporarily as well as permanently. Several approaches are available for gene delivery, such as the use of natural vectors, like modified viruses, or artificial vectors, in the form of liposomes or pep-tidic complexes. Among the key considerations for designing NPs to effectively overcome biological barriers are their physicochemical properties and effects they produce in the living organism. This chapter discusses how the properties of nanocarriers can affect biological responses as well as the functionality of drug and gene delivery systems.

1.2 Types of Nanocarriers/Nanoparticles

NPs can be classified following several parameters, size being one of the first established. In this way, NPs are defined as particles with a diameter <100 nm, but in practical applications it is common for larger particles to be used (up to 1000 nm), especially for drug and gene delivery. NPs can be differentiated based on composition, structure or properties; however, given their heterogeneity it is difficult to talk about pure types. Cargoes can interact with NPs either covalently, non-covalently via weak forces such as electrostatic or hydrophobic interactions, by hydrogen bonding, or can be physically entrapped in the matrix. The various types of NPs described in this chapter are summarized in Figure 1.1.

1.2.1 Viral Nanoparticles (VNPs)

Viruses are infectious nano-sized pathogens (10-200 nm) that naturally deliver their genetic material into a determinate cell line or organ. Viruses are mainly composed of proteinaceous materials (capsid) that have provided the basis for the development of drug and gene nanocarriers. Viral-like NPs (VLPs) differ from VNPs by the absence of endogenous genetic material and their inability to replicate or to alter the host genome. VLPs (Figure 1.1 A) can be formed by self-assembly of capsid proteins over a functionalized inorganic NP core into well-characterized monodisperse structures. Capsid proteins can be engineered and modified with different chemicals and proteins to promote specific targeting and improve penetration properties in VNPs/VLPs. Gene delivery is one of the most studied fields of VNPs, since viruses are natural agents that transfect their genetic material into cells. Although RNA and DNA viruses are potential candidates for gene delivery, they usually present problems of toxicity and immunogenicity, with limitations in the types of transportable cargo. VNP/VLPs have been used to improve vaccine effectiveness due to the strong immune response that is produced in the host. The development of artificial gene vectors such as polymeric NPs or liposomes has attracted the attention of researchers as an alternative to natural viruses, which can produce harmful side-effects when introduced into living organisms.

1.2.2 Micelles and Liposomes

In general, transport of hydrophilic compounds is more favorable in biological aqueous conditions. Hydrophobic cargoes can be transported by amphipathic NPs forming the classical core–shell carriers. For example, in micelles (Figure 1.1B), the hydrophilic part is exposed to medium while forming a hydrophobic core. Amphipathic interactions can produce NPs with several layers, the transport of hydrophilic cargoes in liposomes being possible (Figure 1.1C). Liposomes can easily penetrate the cyto-plasmic membrane and promote cellular uptake. Micellization of chemotherapeutic agents can reduce their cytotoxic effect and increase their effectiveness. Liposomes formed with cationic lipids are extensively used in gene delivery, being one of the most efficient strategies. Cationic lipids can spontaneously combine and condense negatively charged DNA molecules, thus allowing penetration of DNA into cells while being protected from nuclease attack. Although liposomes have high loading capacity, their low stability and strong interaction with cargo can produce a non-stable release. In vivo employment of cationic lipids is limited by their high cytotoxicity and their unspecific absorption by phagocytic cells of the reticulo-endothelial system. A drawback of liposomes is their low stability, which can result in fusion, aggregation, or leakage of the encapsulated drug substance during storage. An alternative to increase the stability of lipid NPs is the use of solid lipid NPs that mainly consist of solid lipid stabilized with surfactants. These NPs show good physical stability and bio-compatibility, although, similar to other hydrophobic NPs, they have a short half-life in vivo and are removed from the blood circulation by the reticulo-endothelial system, particularly in the liver and the spleen.

1.2.3 Polymeric Nanoparticles

Polymeric NPs consist of non-biodegradable and/or biodegradable polymers. Biodegradable polymers, which are widely used for drug delivery, can be divided into two groups, namely biopolymers (protein, peptide and polysaccharide) and synthetic polymers [poly(lactic acid) (PLA) and poly (ε-carpolactone) (PCL)]. Polymer composition and physical properties are factors that influence the effectiveness of these NPs. The high versatility of these NPs, produced by the precipitation of linear polymers into colloidal nanoparticles solution, is due to the countless numbers of available polymers and their combinations (Figure 1.1F).

The more commonly used non-degradable synthetic polymers are N-(2-hydroxypropyl)-methacrylamide copolymer (HPMA), poly(vinylpirrolidone) (PVP) and polyethylene glycol (PEG), since they do not induce a significant cytotoxicity within biological systems. Poly(N-isopropylacrylamide) [poly(NIPAAm)] is a thermosensitive polymer and exhibits a low critical solution temperature at body temperature. These characteristics allow poly(NIPAAm) to be employed extensively as a drug carrier for thermally controlled release. Natural polymers including albumin, silk, chitosan, and heparin have been employed for the delivery of oligonucleotides, proteins, and drugs. A substantial part of the studies on polymeric NPs focus on the encapsulation of larger molecules like DNA or proteins rather than drugs. Nanospheres (Figure 1.1E) are NPs in which the cargo is dispersed through the polymeric matrix. Biodegradable nanospheres are especially suitable for the controlled release of drugs,' because the choice of polymer for NP formation promotes a different degradation rate under selected conditions. Biodegradable polymeric NPs comprise PLA, poly(glycolic acid), poly (lactic-glycolic acid) (PLGA), poly(methyl methacry-late) (PMMA), or poly(L-glutamic acid) (PGA). These NPs are advantageous because they can undergo hydrolysis to form biodegradable metabolites in biological systems. For applications that require a long-term biocompatible stay in the host organism, PCL can be used for NP synthesis due to its slower degradation rate in comparison with other biodegradable NPs. Polycationic polymers such as poly(L-lysine) (PLL) or linear poly(ethylenimine) (PEI) have been employed for the condensation of DNA to form poly-ion complexes. PLL has been shown to mediate gene transfer by compacting pDNA into a tight toroid structure of ~100nm and rendering it resistant to DNase digestion. Even so, the high cationic nature of PLL and PEI produces cytotoxicity and triggered an immune response by the activation of complement. Another cationic polymer is the natural polysaccharide chitosan, which has shown positive attributes of bio-compatibility and degradability. This linear polymer, which is a soluble derivative of chitin (the main compound of arthropod shells), is composed of randomly distributed D-glucosamine and JV-acetyl-D-glucosamine. Chitosan NPs have the ability to adhere to mucosal surfaces and penetrate into cells; the presence of hydroxyl and amine groups allows chemical modification to increase its bioactivity. For all the above, chitosan derivatives have been studied as non-viral vectors, since its cationic charges allows complexation with DNA or RNA. Cyclodextrins are cyclic oligosaccharides with a lipophilic inner cavity and a hydrophilic outer surface. Their amphipathic nature allows formation of non-covalent inclusion complexes with drugs, although inorganic compounds are not generally suitable for complexation. These versatile molecules have been employed to increase the loading capacity of NPs, since they are able to enhance the number of hydrophobic sites in NPs structure. Cyclodextrins can mask drug cytotoxicity and even form the backbone of more complex structures for the transport of genetic materials.

1.2.4 Dendritic Nanoparticles

Dendrimers are radially hyperbranched polymers with regular repeat units. They are attractive systems for drug delivery due to their highly defined dispersity, nanometer size range, spheroid-like shape and multi-functionality. Aside from their ease of preparation, dendrimers have multiple copies of functional groups on the molecular surface which enables derivatization for biological recognition processes. Even so, dendrimers are reported to cause hematological toxicity, especially in the case of non-functionalization. Examples of typical dendrimers are poly(propyleneimine) (PPI), poly(amido amine) (PAMAM), poly(2,2-bis(hydroxymethyl)propionic acid (bis-MPA), poly(glycerol-succinic acid) (PGLSA-OH) or epsilon derivatives of PLL. Although their high charge density allows easy insertion into membranes, and can facilitate endosomal escape, dendrimers have low water solubility and exhibit elevated cytotoxicity. For example, PAMAM NPs have limited applications in medicine due to their original toxi-city. PAMAM is known to induce nephrotoxicity as well as hepato-toxicity, and its cationic charge can cause platelet aggregation by disruption of membrane integrity. Nevertheless, it is possible to reduce the cytotoxicity by chemical modification and the combination of different polymeric ends. Branched PEI is one of the most studied and commonly used branched polycationic polymers for gene delivery, due to its cationic charges and lower cytotoxicity compared to PAMAM, although its cytotoxic effect is higher than other cationic polymers. In general, the buffering capacity of polyamines promotes endosomal escape of NPs, since osmotic swelling occurs by the accumulation of chloride ions in the endosome.

1.2.5 Peptidic Nanoparticles

Peptidic NPs are based on the use of peptide sequences that promote cellular internalization known as cell-penetrating peptides (CPPs) or protein trans-duction domains. CPPs are short peptides (6-30 aa) that are able to cross the cellular membrane for intracellular trafficking of cargoes. It has been postulated that the ability of CPPs to be internalized by cells is related to their strong affinity for lipid bilayers. CPP sequences are based on natural protein-transduction domains such as transactivator of transcription of human immunodeficiency virus (HIV-1 TAT peptide), or penetratin (pAnt), which is derived from the third helix of the Drosophila antennapedia homeodomain. Low molecular weight protamine (LMWP) is derived from the natural protein protamide, an arginine-rich nuclear protein that replaces histones during spermatogenesis. LMWP has demonstrated comparable performance to TAT peptide for cellular translocation while being neither as antigenic, mutagenic nor cytotoxic as other cationic peptides. Similar to other NPs, CPPs can be covalently linked to the cargo, forming a conjugate that promotes transport and internalization of the complex via cellular pathways, but this covalent modification may alter the biological activity of cargoes. To circumvent this limitation, a non-covalent strategy for the attachment of cargo without the necessity of chemical cross-linking or modification is preferred. The presence of cationic amino acids such as lysine or arginine in CPPs seems to be one of the factors that help to improve their transfection efficiency. Meanwhile histidine-rich regions can enhance endosomal escape through the pH buffering, or proton sponge effect. In general, cationic CPPs can form complexes with negatively charged DNA molecules based on electrostatic interactions. Besides CPPs, cationic peptides have been used for gene delivery, and consist of consecutive basic amino acid sequences, which compact DNA into spherical complexes, or chromatin-like components such as histories or protamine, which compact DNA in a structured manner. In this way, oligo-arginine has demonstrated similar characteristics to CPP in cell translocation, being superior to other polycationic homo-polymers. The peptide motive Arg-Gly-Asp (RGD) is able to recognize and bind to the αvβ3 /αvβ5 integrins that are expressed in certain cell types such as endothelial cells, osteoclasts, macrophages, and platelets. Integrins are transmembrane glycoproteins that interact with the cellular matrix and promote receptor-mediated endocytosis. αvβ3/α vβ5 integrins are over-expressed in angiogenic endothelial cells, also being suitable markers for neoplasms. Modifications to increase the specificity of NPs for targeted delivery to a specific organelle within a cell include the use of signal peptides such as nuclear localization signals or mitochondrial-targeting peptides. NPs composed of amphipathic peptides can also be used for the delivery of hydrophobic drugs.

1.2.6 Nanocrystals and Nanosuspensions

Nanocrystals are associations of molecules in a crystalline form, composed of pure drug with only a thin coating of surfactants (Figure 1.11). Drug nanocrystals can be generated by "bottom-up" (intermolecular association) or "top-down" (milling of crystals) technologies. Nanocrystals NPs are composed of 100% drug without the addition of carrier materials such as polymeric NPs. Nanocrystals have been more studied for material science than for drug delivery, given that not all therapeutic compounds can be easily crystallized. However, they are the usual choice for the oral administration of drugs, since their nano-scale size improves drug solubility and dissolution rate as well as increasing adhesion to the intestinal wall and capillary uptake. Nanocrystals have also been successfully employed in the parenteral delivery of compounds such as antibiotics or insulin.

1.2.7 Metallic Nanoparticles

Metallic NPs (Figure 1.1J) are heavily utilized in biomedical sciences because they can be prepared and surface-functionalized in many different ways. These NPs can be used in diagnostics as well as for drug and gene delivery. Metallic NPs can be easily synthesized over a broad range of sizes and shapes, and are usually composed of gold, platinum, titanium dioxide, copper, iron oxides [as magnetite (MxFe3-xO4, M = Mn, Ni, Co, Fe) or maghemite (Fe2O3)] and can be functionalized via thiol-metal chemistry. Metallic NPs can combine properties as surface plasmon resonance, magnetism, or anti-oxidant capabilities. Among the most used are the colloidal gold NPs, given that intake gold NPs without apparent cytotoxicity. Shelling NPs with gold will reduce their cytotoxicity while increasing their stability. Gold NPs are one of the most successful inorganic carriers in oncology, where they have shown applicability as drug carriers and in the thermal ablation of tumors. However, the gold NPs with quantum sizes (1.5 nm diameter) can be toxic, because of their ability to penetrate into the cellular nucleus and bind irreversibly to DNA. Moreover, metallic NPs have shown the production of reactive oxygen species (ROS) and oxidative stress, although this effect has been shown ubiquitously in several types of NPs. The safety of the use of metallic NPs in vivo is under debate, considering that divalent cations and heavy metals are toxic.

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Excerpted from Nanomedicines by Martin Braddock. Copyright © 2016 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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