Emerging areas of bone repair materials

Emerging areas of bone repair materials: nucleic acid therapy and drug delivery

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Phil Chambers1, Helen O. McCarthy1, Nicholas J. Dunne1,2,3,4,5 1School of Pharmacy, Queen’s University of Belfast, Belfast, United Kingdom; 2Centre for Medical Engineering Research, School of Mechanical and Manufacturing Engineering, Dublin City University, Dublin, Ireland; 3Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland; 4Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland; 5School of Mechanical and Manufacturing Engineering of Ireland, Dublin City University, Dublin, Ireland 

16.1  Why nucleic acids for bone repair Within this section an overview of why nucleic acids could be the future of bone repair, the bone microenvironment is also considered. Many second-generation bone repair therapies (such as polymer and ceramic scaffolds) have exhibited successful bone formation and biomineralization, the newest generation of therapy options aim to further enhance the osteoinductive potential of these materials through strict biomimicry of the bone tissue microenvironment, resulting in a more defined regulation of osteoprogenitor cell function and homeostasis. Essential to this microenvironment are the inclusion of stimuli that promote favorable cellular response, or activation of specific genes, increasing the osteoinductivity of the material. A combination of biochemical stimuli (addition of biomolecules, surface functionalisation of the material, etc.) and mechanical stimuli (such as three-dimensional (3D) porous structures to promote cell infiltration, attachment, and proliferation) are usually implemented in third-generation therapies to fulfil these requirements. Historically, most research in tissue engineering has tended to focus primarily on the material properties of bone regeneration scaffolds, and this alone is not enough to mimic the in vivo microenvironment. More recently, the importance of cellular interplay and response has become apparent. Most studies now include biomaterials cultured with one type of cell, usually mesenchymal stem cells (MSCs) or osteoblasts, but in recent years focus has turned to coculture systems and/or the use of gene therapy approaches in an aim to better understand the in vivo process of bone formation. In an attempt to promote vascularization of newly formed bone, Buschmann et al. ([2007]) utilized a coculture system of human osteoblasts (hOB) and human endothelial cells (hEC) on a highly porous poly(ester urethane) foam. This coculture scaffold was compared to control groups containing (1) only hOB cells or (2) only hEC cells. Bone Repair Biomaterials. https://doi.org/10.1016/B978-0-08-102451-5.00016-0 Copyright © 2019 Elsevier Ltd. All rights reserved.

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After culture for 7 days in vitro and 15 days ex vivo the scaffold containing the coculture showed vastly improved cell proliferation, 3D scaffold invasion, extracellular matrix (ECM) formation, and blood perfusion exhibiting clearly the benefits of cocultured systems on bone regeneration [1]. Cocultured systems appear to benefit from cellular cross-talk and as previous investigations have shown that the paracrine effects of MSCs include stimulation of angiogenesis, apoptotic protection, the recruitment of host MSCs and other progenitor cells in addition, to stimulation of proliferation and differentiation [2,3]. Furthermore, MSCs transplanted into mice with a stabilized tibial fracture have been found to migrate to the fracture site, integrate within the callus and secrete BMP-2 to aid with the fracture healing [4]. Therefore, the signals and secretions of cells cultured on a bone regeneration scaffold are extremely important, and the cross-talk between cell types will affect the efficacy and quality of any therapeutic treatment. To this end, gene therapy strategies (such as the intracellular delivery of nucleic acids) are gaining momentum for the purposes of bone regeneration. In gene therapy delivery, cells seeded on the bone regeneration scaffolds are used as bioactive carriers of osteoinductive genes facilitating sustained local delivery, allowing these osteogenic factors to be expressed not only for sustained periods, but in more physiological forms and concentrations (rather than the supraphysiological concentrations required for recombinant growth factor delivery) [5]. As a consequence, this has been shown to direct increased natural bone formation [6] and the utilization of genetically altered proosteoinductive cells within a bone regeneration scaffold have exhibited superior performance in a range of animal models [7]. Most gene therapy approaches for bone formation have focused on the use of genes within the bone morphogenetic protein (BMP) family, in order to induce the subsequent osteogenic cascade [7–10]. However, a common issue exists, which is that a suitable and efficient delivery system for such gene therapies is required. There are numerous physical and biological barriers to be overcome for successful nucleic acid delivery for an intended therapeutic effect. Current methods employed to overcome this issue include viral (such as Adenovirus, Retrovirus, Lentivirus), nonviral (plasmids, cationic polymers, liposomes), and cell mediated gene transfer [6,11].

16.2  The biological barriers to nucleic acid delivery systems in the bone environment Such biological barriers include protection of the cargo from degradation in-situ, entry into the cells, disruption from the endosome, release of the nucleic acid payload into the cytoplasm and finally active transport into the nucleus for DNA. For the purposes of nucleic delivery, the treatment efficacy is highly dependent on the successful transport of a chosen therapy to the target-specific cells. A need exists for a suitable delivery vehicle to protect the cargo from degradation, facilitate cellular membrane penetration, accumulation at the required sites, and successful transfection

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of the target cells. Historically, the safest and easiest method is the incorporation of naked DNA [12], however, in vivo gene delivery using this method has continually shown poor efficiency requiring large amounts of DNA, and any attempts to increase efficiency to a level that would allow clinical use have so far been impossible [13]. The introduction of physical methods to counteract the poor efficacy of delivering naked DNA such as ballistic gene delivery, electroporation, sonoporation, and microinjection [14] have increased the ex vivo applications, but in vivo efficacy still remains poor. Therefore, if the delivery of nucleic acids is to be a successful clinical platform technology in bone tissue regeneration, a suitable vehicle for the efficient delivery and promotion of osteogenesis must be found. The human body employs a host of sophisticated defense mechanisms to protect cells from the invasion of foreign material and therefore many hurdles must be overcome for the successful transfer of nucleic acids to the intracellular region for the intended effect. Some of the most relevant barriers to cellular uptake are described below.

16.2.1  Extracellular barriers 16.2.1.1   Skin The first barrier that any therapy must overcome is the skin, specifically the epidermis. The epidermis functions as a physical barrier to the external environment, protecting from infection and modulating water transfer between the external and internal environments [15]. Therefore, the only way to penetrate this barrier is with use of physical methods like those described previously (microneedles, ballistic gene delivery etc.), with the use of ex vivo scaffolds [16], or by utilizing systematic delivery of the osteogenic factors, usually via an implant for the specific case of bone regeneration.

16.2.1.2  Mononuclear phagocyte system The mononuclear phagocyte system (MPS), also known as the reticuloendothelial system, consists of phagocytic cells employed by the immune system for the identification and removal of foreign material. In the environment of bone repair, osteoclasts in the bone tissue, and monocytes in the bone marrow and blood, are the specific cell types that phagocytose any foreign biomolecules present [17]. The MPS binds serum proteins known as opsonins to the surface of foreign molecules, in a process known as opsonization [18]. Opsonization essentially “flags” the material for phagocytes to being the process of engulfment and subsequent digestion. Therefore, any new therapy for bone regeneration must exhibit the necessary physiochemical and morphological characteristics to avoid detection by the MPS. An increase in opsonization is found with nanoparticle (NP) and gene delivery systems with a cationic charge (increasing the likelihood of binding to the anionic serum proteins). This can be an issue as most cell-targeting delivery systems tend to be cationic in nature to facilitate increased binding to the proteoglycans on the cell membranes. For this reason, the surface charge of any therapeutic agents must be optimized for each application, to ensure that the cell-targeting nature of the molecules is not affected, whilst also ensuring that the

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increased cationic charge will not result in opsonization. The morphological characteristics are also important to evade detection by the MPS. It has been reported that particles <150 nm in size [19], and spherical in shape [20], experience a decrease in serum protein binding. The rationale for this effect is that NPs in this size range are seen to be similar in size to the serum proteins, and as a consequence, the potential contact area between the two is minimized; that is, neither the particle nor the serum protein has enough contact points to facilitate binding [21]. Many delivery systems utilize extra methods to avoid detection by the MPS including PEGylation and biomimetic surface coatings, such as using autologous erythrocytes/leukocytes or “selflike” peptides [22–24]. However, the addition of surface moieties has a detrimental effect on the efficient delivery of cargo (explained further in Cationic liposomes section), and must be carefully balanced for optimal therapeutic efficacy.

16.2.1.3  Extracellular matrix (ECM) and the interstitial space Another major barrier to the targeting of cells within the bone microenvironment is to cross the interstitium or ECM. The ECM is comprised of a cross-linked network of collagen and elastin fibers, proteoglycans and hyaluronic acid [25], and is responsible for the transport of oxygen and other supportive nutrients for the proliferation and maintenance of cells, in addition, to providing the structural integrity required for bone. A highly developed ECM may prove to exhibit significant resistance to the diffusion of therapeutic cargo, causing therapies to be delivered an unwanted distance away from the intended target. The spaces within the ECM are filled with interstitial fluid (ISF), forming a hydrophilic gel [26]. A pressure gradient within the ISF is present as a means for active diffusion and transport of molecules throughout the interstitial space. Therefore, any delivered therapeutic agent must remain stable in the presence of the ISF and the associated pressure gradient and fluid flow. Surface modifications or coatings to intended deliverables (such as the use of superparamagnetic NPs) [27], as well as adhering to strict morphological characteristics are methods currently utilized to aid the delivery of therapeutic agents through the interstitial space and ECM.

16.2.1.4   Phospholipid bilayer The last barrier for a therapeutic agent to overcome on its journey to the inside of a cell is the phospholipid bilayer. This semipermeable cell membrane consists of a structured dual layer of lipid molecules responsible for the mediation of molecule diffusion from the extracellular to intracellular regions (or vice versa). Cellular uptake from the extracellular region usually occurs via two main mechanisms: (1) endocytosis or (2) direct translocation [28].

Endocytosis Endocytosis is a process that occurs naturally in all cell types. The triggering of a cell to take up a molecule via endocytosis can occur via electrostatic interaction with cell surface receptors (proteoglycans), or via direct interaction with the plasma membrane [29]. Endocytosis is the umbrella term for several pathways that are responsible for the cellular

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uptake of “large” particles (0.5–10 μm) via phagocytosis [30], or for smaller particles (<0.5 μm) via pinocytosis (including clathrin or caveolae-mediated internalization). Clathrin-mediated endocytosis refers to the uptake of material via the “packaging” of the cargo within a clathrin-coated vesicle, formation of which is assisted by the budding caused by the GTPase dynamin. The clathrin-coated vesicle is then sorted by early endosomes and either sent back to the surface, or moved to more mature endosomes prior to later compartmentalization in multivesicular bodies or fusion with lysosomes for subsequent acidic degradation [31]. Clathrin-mediated endocytosis only occurs for particles <200 nm in size, due to the observed upper limit on the size of a clathrin-coated vesicle [32], and the rate of uptake via this method also appears to be size-dependent, with particles in the range of 50–100 nm observed to be taken up at a higher rate [30]. As this method of entry involves encapsulation in endosomes, special care must be taken to ensure the required cargo to be delivered is not susceptible to acidic degradation in the early/late endosomal environment with intravascular pH levels ranging from pH 6.5–4.5 [33]. Caveolae-mediated endocytosis is another energy-dependent, but clathrin-independent, pathway of cellular uptake. Caveolae are a subdomain of the well-characterized glycolipid rafts, rich in cholesterol and sphingolipid, the function and maintenance of which is primarily due to caveolin-1 [34]. This method of entry is often exploited by pathogens for direct entry into a cell, or for the assistance of further pathogen entry that are larger than a single caveolae [35]. A single caveolae has dimensions of ∼70 nm [36] consequently is only utilized to take up particles in this region of size, or smaller. This process of entry is favorable to pathogens due to the nonacidic environment, protecting them from any potential lysosomal degradation. This process of cellular entry, however, is much slower than the clathrin-mediated process, due to the low mobility of caveolae [37]. Although clathrin and caveolae are the most utilized method of endocytosis by cells, any cells that are devoid of clathrin or caveolin-1 utilize a wide range of other methods for cellular uptake (collectively known as clathrin and caveolae-independent endocytosis). Currently many of the mechanisms are poorly understood or yet to be discovered, however, Lundmark et al. [38] were able to identify a highly prevalent method of endocytosis in HeLa cells involving GTPase Regulator Associated with Focal Adhesion Kinase-1 (GRAF1). GRAF1 was observed to regulate the clathrin/ caveolae-independent endocytic pathway known as the CLIC/GEEC pathway. Macropinocytosis is an actin-dependent process initiated by the rearrangement of the actin cytoskeleton by the activation of receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) [39]. This process of actin rearrangement leads to surface membrane ruffles that give rise to large endocytic vacuoles called macropinosomes, which encapsulate the extracellular fluid (ECF) and any solute present. Macropinocytosis is usually employed by cells for the uptake of particle clusters, or for larger macromolecules [40].

Direct translocation Direct translocation is the term given to energy-independent mechanisms employed by cells for the cellular uptake of extracellular molecules, without the use of internal cell internalization machinery, but by the perturbation of the cellular membrane for

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entry. Direct translocation is the main and most relevant method of cellular entry for cell-penetrating peptides (CPPs), amongst other direct intracellular delivery vehicles [28]. Direct translocation can describe multiple methods of entry, of which pore formation, inverted micelle formation and “carpet”-like model are among some of the main methods observed. Pore formation occurs due to interactions between the CPP and the phosphate groups on either side of the phospholipid bilayer once the CPP surface density reaches a certain threshold, leading to a formation of a pore and subsequent potential across the membrane, allowing influx of particles within the cell [41]. Inverted micelle formation occurs due to an initial electrostatic interaction between the CPP and the membrane, causing a disruption and the flip-flopping of phospholipids, leading to the formation of an inverted micellar structure. This structure permits the peptide to remain trapped within a hydrophilic environment, and the micelle is then capable of moving across the bilayer to the other side, where it then releases the CPP into the cytosol [42]. The “carpet” model of direct penetration describes the process whereby a cationic peptide binds to the negatively charged phospholipids, resulting in a “carpeting” of the cellular membrane. Above a threshold concentration, the CPP disrupts the lipid packing, facilitating the internalization of the CPP [42]. Although distinction has been made between the various methods of traversing the cellular membrane (clathrin/caveolae endocytosis, direct penetration, etc.), in reality a combination of entry methods are utilized simultaneously. For this reason, it is important to select a delivery vehicle that can be tailored to a predictable method of cellular uptake, as the fate of the cargo will vary depending on the method of internalization. As described previously, physical characteristics such as particle size, are also reported to play an important part in the internalization fate. Other characteristics that can have an effect include particle charge, aspect ratio and topography, as well as the specific internalization characteristics of the cell, which can vary between cell types.

16.2.2  Intracellular barriers 16.2.2.1   Endosomal escape The endocytic pathway is the most predominantly utilized uptake mechanism, and a membrane bound vesicle known as an endosome will engulf any cargo. If the cargo cannot escape during the early stages of endosomal maturation, it will be destined for lysosomal degradation and secretion by the cell. The cargo must be stable at pH 6.5–6.0 in the early endosome and at pH 5.5–4.5 in the mature late endosome [33]. Any cargo contained within the endosome must escape at this late stage, before the endosome fuses with the digestive enzyme-containing lysosomal vesicle. Failure to overcome this hurdle leads to a very low level of therapeutic delivery to intracellular targets. For these reasons, many therapeutic agents with promising in vitro promise fail to realize a potential in in vivo applications due to the lack of bioavailability. While many viral-based delivery systems have displayed efficient endosomal escape both in vitro and in vivo [43,44], endosomal escape is still a rate-limiting factor in

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nonviral delivery systems. Many methods have been investigated for the combating of this issue, and one of the most promising methods involves the use of fusogenic peptides. A fusogenic peptide will undergo conversion from a β-sheet structure at the physiological pH 7.4 to conform to an α-helical structure at the acidic endosomal pH. The helical structure facilitates fusion to the endosomal membrane, allowing the escape of any endosomal inclusions [45]. Other methods of endosomal escape utilized by therapeutic delivery vehicles include pore formation by cationic amphiphilic peptides (AMPs) and membrane rupture due to the “proton sponge effect” caused by protonation [46]. Protonation can be exploited in the lower pH of the endosomal environment. Typically, these delivery systems have a high buffering capacity that swell under protonation, and examples of such delivery systems include cationic polymers and CPPs. The acidic endosomal pH causes protonation leading to the inflow of H+, Cl− and water into the endosomes, resulting in osmotic swelling and eventual rupture of the endosome.

16.2.2.2   Cytoplasm Following endosomal escape, therapeutic agents introduced within the cell are now located within the cytoplasm. The cytoplasm is a densely packed region full of cellular organelles (such as mitochondria, endoplasmic reticulum, Golgi apparatus) and cytosolic components (cytoskeletal filaments, soluble macromolecules such as proteins) [47], and as such is molecularly crowded. Therefore, transport of any delivered cargo within the cytoplasm that is intended for nuclear localization is severely hindered. For gene therapy, the cytoplasm is an inhospitable environment for DNA due to low transport mobility coupled with the presence of nucleases which will degrade free DNA. Lechardeur et al. [48] demonstrated that free DNA was degraded in the cytoplasm of HeLa and COS cells, exhibiting a half-life of only 50–90 min. This poses a significant barrier to the delivery of naked DNA, or DNA complexes that tend to disassociate in the cytoplasm before reaching the nucleus. Recently, studies have found the limiting factor in nucleic acid mobility through the cytoplasm is the size and spherical nature of the molecule, with circular plasmid DNA (pDNA) exhibiting faster transport than the equivalent linear structures [49]. There can only be two mechanisms by which cytoplasmic pDNA is transported to the nucleus; either by (1) diffusion or (2) active transport. However, due to the crowded nature of the cytoplasm, and the size of the DNA, it is highly unlikely that diffusion is the main transport mechanism. A study by Lukacs et al. [50] demonstrated that mobility of DNA larger than 250 bps inside the cytoplasm of cells was significantly slowed (>17-fold) compared to mobility in water, and that DNA larger than 2000 bp was simply not able to diffuse in a timeframe that is considered physiologically viable. It is thought that the high concentration of actin filaments, tubules and other microstructural cellular components are a major retardant to the diffusion of DNA in the cytoplasm. The highly cross-linked nature of the actin cytoskeleton was observed to be a major limiting factor, as the size-dependent diffusion rate of DNA appeared to no longer apply when the actin network was disrupted with the use of cytochalasin D [51]. This observation makes physical sense, as previous studies have reported that actin filaments tend to be approximately 100–500 nm

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in length, with the spacing between filaments observed to be ∼100 nm, and a 250 bp fragment of double-stranded DNA would exhibit a linear size of around 85 nm [52]. Therefore, it is easy to see why DNA may experience diffusional hindrance in the cytoplasmic environment. Consequently, since it is possible to show successful nuclear transfection with DNA it is assumed that the main mechanism of nuclear localization is by active transport along the cytoskeletal elements. Many viruses have been shown to utilize such mechanisms efficiently, such as the herpes simplex virus (HSV), which has been shown to shuttle along the microtubule elements in the cytoplasm of HeLa cells, moving from the periphery of the cell toward the nucleus with the molecular motor dynein implicated for such movement [53,54]. Further studies have shown that depolymerization of the microtubule network in HeLa cells using an actin depolymerization agent (nocodazole), showed a two-fold decrease in infection by HSV, further supporting the importance of cytoskeletal trafficking of complexes [55].

16.2.3  Suitable vehicles for the efficient delivery of therapeutic agents As described in Sections 16.1 and 16.2, many hurdles must be overcome for the successful intracellular delivery of any therapeutic agents in vivo. Therefore, the design of a suitable delivery vehicle is imperative for the successful delivery of the required cargo to the cytoplasm (in the case of RNAi) or the nucleus (pDNA), avoiding degradation or loss of function in the process. The delivery system must facilitate binding to the therapeutic agent, increase the extent and rate of cellular uptake, protect from degradation during circulation/endocytosis, facilitate endosomal escape, and disassociate from the cargo once cytoplasmic (or nuclear) delivery has occurred. Furthermore, if DNA is the chosen therapeutic agent, nuclear targeting and permeation of the nuclear membrane is one more limiting factor that needs to be overcome, usually achieved with the use of nuclear localization sequences (NLS) or other targeting ligands [56,57].

16.2.3.1  Nonviral versus viral delivery methods Having evolved to enable effective gene delivery into cells, viruses are currently one of the most effective methods of cell transfection, and in hard-to-transfect cells. Recombinant viruses can be generated by the insertion of exogenous DNA with the genome of the virus and is subsequently delivered inside the target cell via infection [58]. A wide variety of viral vectors have been developed to deliver therapeutic agents (most commonly genes), for the transient (adenovirus, vaccinia virus) or stable (retrovirus, lentivirus) transgene expression [59]. Viral delivery vehicles remain the “gold standard” in efficient in vitro and in vivo cellular transfection, and have been the subject of much research, with promising results being reported from various clinical trials [60]. However, due to potentially fatal adverse effects with the use of viral vectors, at this stage the FDA has yet to approve any virus-based therapies. The potential side effects of using viral vectors include: immunogenicity, off-target effects, toxicity, and insertional mutagenesis, including activation or proto-oncogenes and inactivation of tumor suppressor genes [61].

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The inherently dangerous adverse side effects of using viral vectors for the transfection of cells, coupled with the associated expense, has led to extensive research into nonviral delivery methods that are simpler, more predictable, reproducible, and most importantly, provide a safer alternative. Historically, the major limitation on nonviral delivery systems was the observation of low levels of transfection and short-term gene expression observed in host cells in comparison with the viral alternatives. Fortunately, since only brief expression of osteogenic genes is enough for the stimulation of osteogenesis, nonviral delivery methods can be valuable to bone regeneration applications [62]. In addition, many recent studies have been able to exhibit levels of nonviral transfection on par with viral delivery systems [63,64], which points to the exciting potential of nonviral delivery systems. Additionally, such systems were also seen to exhibit much lower levels of toxicity, greater site-specificity, low immunogenicity and an ability to deliver large genes [65] making nonviral delivery systems an attractive and more clinically relevant option for the delivery of therapeutic agents. Some of the most commonly utilized nonviral systems for the delivery of therapeutic agents are described below and put into context for applicability for bone regeneration.

16.2.3.2  Nonviral: physical methods Physical methods used for the intracellular delivery of therapeutic agents rely on the application of external forces to the cellular membranes to increase transient permeability allowing entry of intended deliverables. Physical methods are often selected for use in hard-to-transfect cells, or to supplement another method of delivery. Usually employed in vitro, and rarely used in delivery of miRNAs, physical delivery of therapeutics can damage cell integrity and elicit a level of apoptotic response. Some of the physical methods employed include the use of mechanical, electrical, ultrasonic, hydrodynamic, and laser-based energy [66]. However, for bone regeneration scaffolds the use of physical methods do not present a viable option because of the need for a long term sustained release of therapeutic agents in vivo, which is simply not possible by way of physical intracellular delivery methods.

Microinjection of naked DNA The most simple and obvious method of introducing genes within a cell or tissue is by mechanical injection directly within the required location. Requiring no carrier system, this method of introduction is inherently safe, however, a major limitation of this method exists in the very low levels of efficiency observed [13], in some cases as low as 1% of cells experiencing transgene expression, due to rapid degradation by nucleases in the serum, or clearance by the MPS.

Biolistic delivery (the “gene gun”) Biolistic (a contraction of biological and ballistic) delivery involves the high-speed permeation of cell membrane with the use of a ballistic “gene gun.” Tungsten or gold particles (∼1–3 μm in diameter) are coated with DNA and accelerated using high voltage electric sparks, or a helium discharge [67,68]. This method aimed to overcome the difficulty of delivering naked pDNA by attachment to a high velocity particle carrier.

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Ballistic therapeutic delivery has shown to be effective in the delivery to exposed tissues such as skin (where it can penetrate into the epidermal and dermal layers) and any exposed surgical tissues, and has been particularly effective for use in DNA immunization and vaccination [69], and in the treatment of skin wounds and burns [70]. The efficiency of ballistic DNA delivery depends on the parameters selected, including the amount of DNA used, particle size, timing of the delivery, and the optimal acceleration conditions for the final localization of the DNA-coated particles [14]. Limitations to this method of therapeutic delivery include the slow preparation of particle carriers, often taking up to 2 days to prepare, and large variations in transfection efficiency due to shearing of DNA or particle carrier agglomeration [59]. Inflammation and irritation have also been reported at the introduction site of therapeutics delivered by ballistic means.

Electroporation Electroporation utilizes an electrical field to increase cell permeability, in a phenomenon known as electropermeabilization. First reported by Neuman et al. in 1982 [71], they found that by applying an electrical field greater than the capacitance of the membrane, charges of opposite polarity would arrange on opposing sides of the cell surface. This would create the formation of a pore due to the potential difference seen at this precise point, facilitating cellular entry through the pore. If the cells are to remain viable, the pore can then be closed if the field strength and pulse duration were correct. However, if the pore remains open, this leads to cell death, a method that is often utilized for the destruction of cancerous cells in patients [64]. Drawbacks to the use of electroporation exist in the difficulty in selecting the correct electrical field properties for optimal electroporation of the intended cell or tissue type, as incorrect properties may lead to irreversible cell damage. Furthermore, it has been reported that different conditions are required (field strength, pulse duration, etc.) for the delivery of differing molecules, with size and concentration affecting such properties. However, even with these described drawbacks, electroporation has been shown to be one of the most effective methods of nonviral delivery, and a reliable method of in vivo gene delivery in bone [72], and various other tissue types such as muscle [73], skin [74] and liver [75].

16.2.3.3  Nonviral: chemical methods Chemical carriers for the non-viral delivery can be broadly split into the categories of inorganic nanoparticles, lipid based, polymer based and peptide-based systems [76].

Inorganic nanoparticles Inorganic NPs (iNPs) used for the delivery of therapeutic agents include NPs prepared from ceramics (such as carbonate or phosphate salts of calcium, magnesium, or silicon) or metals (including iron, gold, and silver) [77]. Particles with a complex chemistry in the size range <100 nm can be easily prepared from these inorganic components, which facilitate surface coating for the binding of DNA, or for gene targeting. The nanoscale of iNPs offers various advantages to the delivery of therapeutic agents, including the ability to transfect cells successfully both in vitro and in vivo via

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nonendocytic routes delivering NPs directly to the nucleus avoiding potential degradation in the endosome/lysosome [78]. Qureshi et al. [79] utilized silver nanoparticle complexes for the successful delivery of photoactivated miR-148b mimics, resulting in the osteogenic differentiation of MSCs. The transfection efficiency with the use of iNPs is not as good as other nonviral transfection options, however, iNPs are able to escape from the MPS, are inert to immune response due to the bio-inert chemistry, exhibit low toxicity and possess good storage stability. It must be noted that if DNA is to be delivered with the use of iNPs, by binding to the surface of the NP, further protection from intracellular degradation by nucleases is necessary. The use of superparamagnetic based iNPs (such as iron oxide NPs) are also of major interest for therapeutic delivery, as they are responsive to magnetic fields, facilitating the targeted delivery of cargo via magnetic field manipulation [80].

Cationic liposomes Cationic liposomes are synthetic lipid spheres that are composed by 1 or 2 fatty acids and alkyl functional groups, in addition, to a positively charged head group and hydrophobic tail group, connected via a linker structure. Cationic lipids possess the ability to form unilamellar vesicles, often with the help of a neutral or helper lipid, such as dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC) [81,82], and these vesicles are capable of spontaneous interaction with DNA. The positively charged group on the cationic lipid interacts with the negatively charge phosphate groups on the DNA, resulting in self-assembly of lipid-DNA complexes known as a lipoplex. The delivery of genetic material within a cell with the use of liposomes (lipofection) is a process that was first pioneered in 1987 by Felgner et al. [83], but is now an intense area of interest for the nonviral delivery of therapeutic agents, and is currently one the most extensively investigated and commonly used delivery systems [84]. Liposomal delivery has been shown to be a highly effective method of gene transfer, and for bone regeneration in particular Park et al. [7] were able to report a comparable level of bone regeneration in critical-size defects of rat mandible treated with liposomal gene delivery to that by adenoviral vectors, negating the safety issues and constraints of requiring viral delivery. Cationic liposomes are more commonly used than anionic or neutral liposomes due to ease of production, high affinity for the negatively charged glycoproteins and proteoglycans of cell membranes facilitating cellular uptake, and a nonpathogenic and nonimmunogenic response. However, anionic and neutral liposomes are often utilized in the delivery of miRNAs, due to the lack of stability of cationic liposomes in circulation. Cationic liposomes suffer from unspecific binding to serum proteins, causing accumulation and subsequent removal by the MPS. For example, Huang et al. [85] were able to successfully increase miR29b expression in acute myeloid leukemia via miRNA delivery in anionic lipoplexes, and similarly, miR-34a transfer in lymphoma has been achieved through the use of neutral lipids [86]. The stability of cationic liposomes can be increased with the use of surface modifications, such as conjugation with polyethylene glycol (PEGylation), to create so-called “stealth liposomes” that are capable of avoiding detection by the MPS, consequently increasing circulation time [87,88]. It should be noted however, additions of PEG moieties to the liposome are associated with a decrease in interaction

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between the liposome and cellular membrane, leading to a decrease in levels of transfection [89]. Although the stability of cationic liposomes can be an issue, the main drawback with liposomal delivery is the acute cytotoxicity observed due to the strong cationic nature and lack of linker group degradation [90]. Optimal delivery of DNAlipoplexes has been reported when the positive/negative charge ratio is around 1, and liposomes are observed to become cytotoxic when the ratio of lipid:DNA surpasses 3:1 [64,83]. Liposomes continue to be an area of interest in nonviral delivery, and hundreds of combinations have been designed involving various lipids and surface modifications, including peptides and ligands, to combat stability and toxicity issues, and to offer more control over particle size, morphology, and degradation characteristics [91]. Many commercially available liposome transfection agents are commonly used as a benchmark for transfection efficacy such as Metafectene, Lipofectamine 2000, and Lipofectin [92], and as of August 2016 lipofection accounts for 4.7% (n = 115) of viral and nonviral vectors used in human clinical gene therapy trials worldwide [93].

Cationic polymers Cationic polymers were introduced around the same time as cationic liposomes by Wu et al. in 1988, who successfully utilized a poly-l-lysine (PLL) carrier to deliver pDNA to rats in vivo [94]. Like liposomes, cationic polymers are capable of interacting with DNA material to form a complex known as a polyplex. Further development lead to the second generation of polymeric carriers, the first of which was pioneered by Boussif et al. in 1995 who introduced the use of polyethylenimine (PEI) for the intracellular delivery of DNA [95]. PEI is a dense, highly cationic polymer that exists in either branched (BPEI) or linear (LPEI) forms, both of which are seen to exhibit high transfection activity [95–97] in vitro and moderate activity in vivo. Comparably, LPEI has been observed to exhibit lower toxicity and better gene transfer efficiency than the BPEI counterpart [98]. Although PLL is still the most commonly investigated polymer delivery vector due to the good biodegradability (resulting in lower toxicity) and has been used in a wide range of applications, PEI is considered by many to be the gold standard in polymeric carriers. PEI possesses high transfection efficacies [97], which are attributed to the high density of amine groups, the majority of which are unprotonated at a physiological pH. Upon entry into the cell via endocytosis, and compartmentalization in the endosome, the unprotonated amines exerts the “proton sponge effect,” as described in Section 16.2.1, effectively absorbing protons transported into the intracellular region by the ATPase proton pump, preventing acidification of the endosome [99]. What follows is an influx in chloride ions into the endo-lysosomal vesicle, resulting in osmotic swelling and destabilization of the membrane, preventing subsequent lysosomal degradation of the contained polyplexes. Further intracellular trafficking of DNA delivered by PEI-DNA polyplexes has been observed to result in high levels of transgene expression [100]. Disadvantages of PEI include low levels of biodegradability [101], with subsequent toxicity. The toxicity of PEI in particular has been observed to behave in a dose-dependent manner, and for this reason, the use of this polymer in vivo is severely limited due to the high concentrations necessary for sufficient transfection

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efficacy [102]. Although many PEI-derivatives have exhibited lower toxicity (involving functionalization with other copolymers such as PEG [103]) and higher transfection efficacies (with the inclusion of hydrophobic substituents such as lipids or polycaprolactone, or targeting ligands such as transferrin [104,105]), PEI-based therapeutics are still a long way from use in clinical treatment. Based on a record of proven clinical safety [106], poly(lactic-co-glycolic acid) (PLGA) has been widely investigated as a nontoxic alternative to PEI/PLL, and a suitable carrier of therapeutic cargo. PLGA is a biodegradable polyester that undergoes bulk hydrolysis, facilitating a sustained release of cargo, with the degraded products easily removed by the citric acid (Krebs) cycle [107]. Due to the attractive biodegradability, biocompatibility and ease of processing, FDA-approved PLGA carriers are extensively used in drug delivery [107–110]. In particular, the ability of PLGA to bind to and protect DNA from in vivo degradation, by the rapid escape from the endo-lysosome, has led to the increased use of PLGA polyplexes for the delivery of nucleic acids in addition, to proteins and peptides [110–113]. PLGAcontaining drug delivery devices are routinely in clinical use, such as Lupron Depot, utilized in the treatment of prostate cancer [114]. Although safer than other cationic polymeric carriers, conventional PLGA contains negative charges which severely hinder the encapsulation of anionic cargo (such as DNA) and reduce overall transfection efficacy. It is therefore necessary to add other copolymers such as chitosan [115,116].

Chitosan Chitosan is a naturally occurring cationic polysaccharide that has proved to be an attractive option for use in the polymeric delivery of therapeutics due to the outstanding biodegradability, excellent biocompatibility, low immunogenicity, antimicrobial activity and ease of production [117,118]. The in vitro transfection efficiency of chitosan has been observed to correlate with such factors as the degree of deacetylation, molecular weight of the chitosan selected, and in the case of DNA-complexing, the ratio of nitrogen atoms in the chitosan to the phosphorus atoms in the DNA (known as the N:P ratio). Therefore, varying efficacy is seen in the intracellular delivery using chitosan depending on the chosen chitosan carrier. For example, high molecular weight (HMW) chitosan produces a more stable particle compared to low molecular weight (LMW) chitosan (including better DNA condensation) but disadvantages include increased aggregation of particles and a low solubility at a physiological pH [119]. LMW chitosan outperforms HMW with respect to intracellular release but the degree of complexation is severely hampered [120]. Chitosan does not have the pH buffering capacity required for endosomal disruption [121]. However, abundant amine and hydroxyl groups contained within chitosan enables simple modifications for increasing transfection efficiency or gene transfer. Typically, chitosan is cografted with other polymers such as PEI or PEG [122,123]. The naturally occurring biocompatibility and biodegradability properties of chitosan make it a highly investigated option for the targeted delivery of therapeutics, but low solubility, poor transfection efficiencies and a lack of targeting are limiting factors.

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Dendrimers Dendrimers are synthetic, homogeneous, monodisperse and spherically symmetrical polymers consisting of a well-defined tree-like branch structure [59]. Dendrimers contain positively charged terminal groups that are capable of functionality for the attachment of antibodies, therapeutic agents, or DNA at a physiological pH, forming dendriplexes, like lipoplexes or polyplexes [124]. Modification of dendrimer terminal groups results in a hydrophilic interior and hydrophobic exterior or vice versa. These characteristics confer flexibility of use [59]. The most common dendrimers (and commercially available) are poly(amidoamine) (PAMAM) and subsequent derivatives, which are observed to form spherical polymers with good aqueous solubility and the amount of positively charged exposed surface groups is an advantage facilitating endo-lysosomal escape [125]. The nanosize (5–20 nm) of PAMAM dendrimers in addition, to high molecular uniformity, ease of preparation, ease of large scale production, and the ability for precise functionalization due to numerous copies of surface groups present, has meant that they are an extensively used in the delivery of therapeutics [126–128]. Oliveira et al. [129] were able to induce osteogenic differentiation in rat bone marrow-derived MSCs (BMSCs) via scaffold-mediated delivery of dexamethasone loaded PAMAM dendrimers. While Santos et al. [130] used dendrimers to deliver a plasmid encoding BMP-2 inducing osteogenic differentiation in rat BMSCs. The structure of a dendrimer starts with a core molecule that is subsequently grown through a stepwise polymerization process. With each new layer (known as a generation G1, G2, G3, etc.), the number of surface amine groups is observed to increase, as is the MW and subsequent charge density [131]. The transfection efficiency and toxicity of dendrimers is generation-dependent. For example, PAMAM dendrimers G0–G3 have been reported to exhibit poor gene transfection efficiency and a low cytotoxicity, but higher generations such as G4–G8 exhibit a better gene transfection but with an associated increase in toxicity [132]. This is similar to the optimization trade-off between LMW and HMW chitosan polymer carriers, as described in Chitosan section. As the ideal transfection vector requires the highest possible efficiency and the lowest possible toxicity, many researchers are working to optimize the properties of PAMAM dendrimers for therapeutic delivery [133,134].

Cell-penetrating peptides Cationic peptide-based vectors are generally considered to hold many advantages over the various other options of nonviral delivery and have been shown to complex anionic cargoes, target specific intracellular or extracellular receptors, disrupt endosomal membranes, and protect the cargo in the cytoplasmic environment [135]. Additionally, peptide sequences have been conjugated to lipoplexes, polyplexes, or dendriplexes, to target or improve efficacy [136,137]. CPPs are a unique class of fusogenic peptide sequences designed to interact with the endosomal membrane, facilitating escape and intracellular delivery of therapeutic agents. CPPs rose to prominence in the late 1980s following multiple reports that the transactivator of transcription (Tat) protein of the human immunodeficiency virus

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(HIV) was able to efficiently enter tissue-cultured cells and promote viral gene expression [138,139]. Generally no more than 30 amino acids in length, most known CPPs exhibit a net positive charge at physiological pH (ideal for the condensation of DNA) and normally contain a high content of basic residues, ranging from 17% in human calcitonin and its C-terminal fragment 9–32 (hCT(9–32)) [140] to 100% with polyarginines [141]. It is generally accepted that CPPs can be placed into one of three major classifications; protein-derived CPPs, chimeric CPPs, or synthetic CPPs (Table 16.1) [28]. Protein-derived peptides are normally derived from the minimal effective partial sequence of the parent translocation protein that interacts with cell membranes, known as protein transduction domains. Chimeric peptides usually involve the fusion of hydrophilic and hydrophobic domains from other natural sources. Synthetic peptides are rationally designed sequences that aim to create well-defined α-helical structures for amphipathicity, usually by involving motifs of other known CPPs. With the exception of polycationic homopolymers such as polyarginines or polylisines, most CPPs exhibit a distinct amphipathic structure (such as the chimeric CPP transportan [146]), or are seen to become amphipathic when conforming to an α-helical structure (such as the synthetic peptide MAP) [148]. CPPs are readily internalized by a wide range of cell lines [142] and primary mammalian cells (such as rat brain and rat spinal cord [142], calf aorta [151], and porcine and human umbilical vein endothelium [148]) for the delivery of a wide array of both large and small cargo such as nanoparticles, proteins, liposomes and DNA [152]. Suh et al. [153] were able to report the efficient intracellular delivery and subsequent upregulation of microRNA-29b into hMSCs with the use of an arginine-rich CPP, and reported a significant increase in osteogenic effect compared to the same miRNA delivered via lipoplexes. Furthermore, the successful in vivo efficacy of CPPs is routinely reported, and is widely used in the delivery of small interfering RNA (siRNA). Intravenous injection of MPG/cyclin-B1 siRNA particles was reported to efficiently block tumor growth [154,155], and the use of a small peptide derived from the rabies virus glycoprotein linked to polyarginine-9 was capable of siRNA delivery to the central nervous system. Although CPPs are successfully utilized for the intracellular delivery of therapeutic cargo, the method of internalization has many conflicting reports. It is thought that CPPs are taken up by cells via the energy-dependent endocytic routes (see Endocytosis section ), however, a large number of investigations report internalization of CPPs under conditions that should otherwise prevent translocation via endocytosis, such as low temperatures (e.g., 0–4°C) or in the presence of various endocytic inhibitors [142–144,146,156]. However, endocytosis does appear to play a role in the uptake of CPPs to a certain extent [157], therefore it is clear that CPPs are not limited by one method of internalization, but more likely use a combination of pathways. Differing CPP sequences have been observed to translocate by different methods, alluding to a dependence on CPP sequence as a potential contributor to the method of internalization. In Tatderived CPPs it has been postulated that endocytosis could be an exclusive method,

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Table 16.1  Commonly utilized cell-penetrating peptides (CPPs) including sequences and origin Classification

Name

Sequence

Origin

Ref.

Protein-derived

Penetratin

RQIKIWFQNRRMKWKK

[142]

Tat pVEC

GRKKRRQRRRPPQC LLIILRRRIRKQAHAHSK

MPG

GALFLGFLGAAGSTMGAWSQPKSKRKV

Transportan

GWTLNSAGYLLGKINLKALAALAKISIL

Pep-1 MAP Polyarganines R6W3

KETWWETWWTEWSQPKKKRKV KLALKLALKALKAALKLA (R)n 6 < n < 12 RRWWRRWRR

Drosophila antennapedia homeodomain (amino acids 43–58) Protein from HIV-1 (amino acids 48–60) Derived from murine vascular endothelial cadherin (amino acids 615–632) Peptide derived from fusion sequence of HIV-1 gp41 protein coupled to peptide derived from the nuclear localization sequence of SV40 T-antigen minimal active part of galanin (amino acids 1–12) coupled to mastoparan via Lys13 HIV-reverse transcriptase/SV40 T-antigen de novo Based on Tat peptide Based on penetratin

Chimeric

Synthetic

[143] [144] [145]

[146]

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[147] [148] [149] [150]

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as Tat 48–60 was observed to not enter live HeLa cells at 4°C, and has been recently thought to enter cells primarily via lipid raft-mediated macropinocytosis [158,159]. Internalization of CPPs as a result of direct penetration is reported as a primary method of entry (see Direct translocation section), with translocation occurring via such as mechanisms as the “carpet” model for pVEC and transportan-10 [160], or via the adoption of an α-helical structure of MAP when in contact with lipid membranes [161] leading to pore formation. It has been reported that Tat-derived CPPs (the most commonly utilized category of CPP) are limited in an ability to escape the endo-­ lysosome, and as such with functionalization of Tat via addition of a histidine tail, Lo et al. [162] were able to improve the gene efficiency more than 7000-fold compared to controls. Histidine is an amino acid that is often included in the peptide sequence of CPPs due to the promotion of the “proton sponge effect” for endo-lysosomal membrane disruption. Endosomal escape of therapeutic cargo delivered by CPP carriers is of the utmost importance to avoid subsequent lysosomal degradation. Many membrane destabilizing sequences can be found naturally, such as the influenza virus haemagglutinin 2 protein (HA-2) [163]. Upon a decrease in pH, the acidic residues contained within this peptide are protonated, facilitating the formation of an α-helical structure. ­PLL-DNA polyplexes conjugated with HA-2-derived peptide sequences have been reported to increase in vitro gene expression from as little as 10-fold, to as much as 10,000-fold increase over PLL-DNA complexes without the addition of the fusogenic peptide [135]. The pH dependent endosomal disruption behavior of this peptide sequence found in nature has subsequently lead to the creation of synthetic alternatives, such as GALA, KALA and H5WYG [164–166]. GALA is a sequence of glutamic acid (E), alanine (A), leucine (L), alanine (A) amino acid repeats (forming EALA sequence), which creates a fusogenic peptide capable of taking on the conformation of a strong α-helical structure at a range of pH levels (including low pH), exhibiting regions of both hydrophilicity and hydrophobicity [164]. At physiological pH, the glutamic acid residues cause a charge repulsion, however, as the pH is lowered (such as in the endosome) these charges are neutralized, allowing adoption of the α-helical shape. At the endosomal pH, GALA is capable of fusing to the bilayer membranes, inducing leakage of phosphatidylcholine vesicles [135]. Despite exhibiting good results in condensing cationic products [167], due to the anionic nature of GALA, it cannot be used as an efficient method of condensing anionic complexes (such as DNA). KALA is a modified version of GALA with the glutamic acid (E) repeats substituted for lysine (K) [165]. With the addition of lysine, it follows that KALA is cationic in nature and can therefore be used to condense anionic complexes. However, the presence of the lysine residues means that the α-helicity of KALA is disrupted as the pH level decreases, causing a decrease in the fusogenic ability, as well as inciting cytotoxic effects of the peptide [165]. With respect to these properties, KALA is therefore not desirable for use in situations where there may be a variation in pH, such as in the endosomal environment. As such, the ideal CPP should exhibit the properties of GALA (ability to hold α-­helicity at a lower pH) but also be similar to KALA with respect to the condensation of anionic entities.

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Arginine Alanine Leucine Glutamic acid Tryptophan Histidine

Figure 16.1  Alpha-helical structure of RALA peptide. The arginine-rich region provides RALA with its hydrophilic nature and facilitates the binding of anionic complexes. The leucine-rich domain provides the hydrophobic region and allows interaction with lipid membranes. The hydrophobic and hydrophilic regions are separated by an alanine-rich region, subsequently giving RALA amphipathic properties. Tryptophan increases stability within the cell and glutamic acid increases solubility at physiological pH.

To this end, McCarthy et al. [168–171] have designed a fusogenic peptide that fits these desired characteristics of a peptide for the condensation and delivery of anionic complexes. Termed RALA, the lysine residues are substituted with arginine (Fig. 16.1). Arginine is found in naturally occurring condensation motifs and has been shown to exhibit superior DNA transfection compared to lysine [172]. RALA demonstrates strong nanoparticle formation of plasmid DNA, and maintain desirable α-helicity at reduced pH (∼pH 5.0) facilitating endosomal escape of NP cargo into the cytosol of cells. This is an important property of any CPP, as the pH in the endosome has been shown to be in the region of pH 5.0–5.5 [172]. In addition, RALA displays an in vitro transfection efficacy on par with commercially available transfection agents such as Lipofectamine, while beneficially exhibiting a superior lower cytotoxicity profile [168]. CPPs, and fusogenic CPPs in particular, are a highly attractive option for the intracellular delivery of oligonucleotides due to reduced cytotoxicity and immunogenicity compared to viral alternatives. CPPs can be designed to be multi-functional, are easily manufactured and scaled up and so it follows that this class of delivery systems are gaining momentum in the drug delivery field. With respect to bone tissue engineering and regeneration applications, there is the potential for the use of oligonucleotides directly delivered via CPPs such as RALA into the cytosol of cells, facilitating an increase in osteogenic differentiation and accelerating the rate of mineralization of osteoprogenitor/osteoblast cells (Fig. 16.2).

Thymine

Guanine

Adenine

Size < 100 nm Charge ~ +20 mV

Cytosine

4 Arginine

Amphipathic cationic peptide (RALA)

RALA/pDNA complex

1 Clathrin/caveolae mediated endocytosis

Nucle

us

pH<5 –

Osteogenic genes Osteogenic differentiation

Cell

3 Cytosolic release

2 Endosomal acidification

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pDNA

Figure 16.2  Endosomal escape of RALA/pDNA NPs. Cationic peptide RALA interacts with anionic plasmid DNA to create net cationic RALA/ pDNA NPs that are internalized via clathrin/caveolae-mediated endocytosis (1). Upon the decrease in pH experienced within the endosomal environment (2), RALA conforms to the alpha-helical shape, facilitating endosomal escape (3), and delivery of the pDNA to the cytosol for an increase in osteogenic genes (4). 429

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16.3  MicroRNA: the future of nucleic acid therapies for bone regeneration MicroRNAs (miRNA) are small single strand, noncoding RNA molecules ­(approximately 22 nucleotides in length) that regulate posttranscriptional expression of messenger RNA (mRNA) and proteins [173]. First discovered in 1993 by Ambros et al. [174], at the time of writing there are more than 2000 mature human m ­ iRNAs ­identified and cataloged [175], and undoubtedly more will be discovered. The power of miRNA therapeutics comes from the fact that a single molecule can target more than 100 genes, and each gene is normally a target of multiple miRNAs. Given that such a vast number of miRNAs have been identified it has been predicted that miRNA is responsible for the influence of anywhere from 30% to 80% of the human genome [176,177]. miRNAs have been shown to possess an important role in cellular ­processes such as differentiation, proliferation and apoptosis [178–180], but also in many ­biological processes such as the pathogenesis of human diseases such as diabetes [181], neurodegenerative diseases [182], cancer development [183] and tissue regeneration [184,185]. More recently, miRNAs have also been shown to participate greatly in osteogenesis, displaying involvement in osteoblast differentiation through the regulation of signaling pathways, either enhancing or inhibiting differentiation via binding to osteogenic transcription factors (TF) [186–188]. Additionally, miRNAs have been identified as regulators of osteoblast and osteoclast behavior and as a result play are involved in the process of bone remodeling and many bone metabolic diseases (such as osteoporosis) [189].

16.3.1  Biogenesis of microRNA The biogenesis of miRNA in mammalian cells is summarized in Fig. 16.3 and begins with RNA polymerase II transcription of a hairpin primary miRNA (pri-miRNA). The pri-miRNA is transcribed from a miRNA coding region which occurs either in the portions of noncoding regions (introns) of protein-coding genes, or as independent transcriptional units [190]. The pri-miRNA is then cropped by the nuclear RNaseIII type endonuclease Drosha, resulting in a double-stranded stem-loop intermediate, called precursor miRNA (pre-miRNA) that is 70 nucleotides in length [191]. Drosha is assisted by the nuclear protein DiGeorge syndrome critical region gene 8 (DGCR8). DGCR8 is responsible for stabilizing Drosha and recognizes the double-stranded RNA structure of the hairpin pri-RNA. It is also thought that DGCR8 is responsible for the correct orientation of Drosha on the pri-RNA [192]. The cleaving of the primiRNA is an important event that predetermines the mature sequence of the miRNA, as well as the substrate for further downstream events [193]. Cleaved pre-miRNA is subsequently exported from the nucleus into the cytoplasm via the transporter complex exportin-5, which not only facilitates nuclear exportation, but also protects pre-miRNA from nuclear digestion [194]. Once in the cytoplasm, premiRNA is once again cleaved by the RNase Type III endonuclease Dicer resulting in a ∼22 base pair (bp) double-stranded miRNA duplex [195]. Subsequently, via binding

Emerging areas of bone repair materials: nucleic acid therapy and drug delivery

Nucleus

RNA polymerase II

431

Cytoplasm

1 Transcription

(A)n

Hairpin pri-miRNA

Drosha DGCR8 (A)n

DNA

3’ UTR of target messenger RNA

2 Cleavage pre-miRNA

5 mRNA pairing

degraded strand Exportin-5 mature strand 3 Nuclear export

miRNA duplex

Risc

pre-miRNA Dicer 4 Cleavage

Figure 16.3  Schematic representation of the biogenesis of miRNA.

with Argonaute (Ago) proteins, one strand of this duplex is selected for degradation (passenger strand), while the other remains as a mature miRNA and is incorporated into the RNA-induced silencing complex (RISC) [196]. The inherent thermodynamic asymmetry of the miRNA duplex is the primary determinant of which strand will be selected to form the mature RNA, that is, the RNA with the 5′ strand that is the least stably bound with the opposite strand is selected [197]. Finally, mature miRNA complexes typically target the 3′ untranslated region (3′UTR) of the mRNA. The miRNA binds via what is known as the “seed” region, located between nucleotides 2–8 on the 5′- end of the miRNA [197], by either complementary or partially complementary base pairing. Once bound, the mature RISC complex controls specific gene expression at a posttranslational level by the recruitment of various protein factors, and degradation of the mRNA [198].

16.3.2  Prominent microRNAs involved in osteogenesis It is well established that MSCs are the source for osteoblast cells recruited for osteogenesis. MSCs are capable of differentiation into chondrocytes, adipocytes, and osteoblasts, determined by a complex array of osteogenic factors and signals received at a posttranscriptional level, of which miRNAs are among some of the most crucial. Since

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Table 16.2  Commonly reported miRNAs involved in osteogenesis

miRNA

Gene target(s)

miR-26a miR-29a miR-29b

SMAD 1, GSKβ, TOB1 RUNX2, COL1A, COL5A TGFβ3, HDAC4, DUSP2, AcvR2b FAK DLX5, Wnt signaling target CDC25 A HOXC8, HOXB7 TGFβ, VEGF, AcvR1b RUNX2, DKK1 RUNX2, FGFR2 GaINT7, CASP3, GSK3β Osterix HDAC5

miR-138 miR-141 miR-196a miR-210 miR-335 miR-338 miR-378 miR-637 miR-2861

Promotion (↑) or inhibition (↓) or miR required for osteogenesis

Ref.

↑/↓ ↑/↓ ↑

[187,199,200] [201–203] [188,203]

↓ ↓

[204,205] [206–208]

↑ ↑ ↑/↓ ↓ ↑ ↓ ↑

[209,210] [211,212] [213,214] [215–217] [218–220] [215,221] [222,223]

the miRNAs are capable of binding to the target sequences in the UTR of mRNAs, causing translational arrest or degradation by the RISC, it is clear miRNA can potentially have a considerable inhibitory or enhancing effect on osteogenesis by binding to osteogenic TFs. Vast numbers of miRNAs have been reported to be involved in the differentiation of BMSCs into mature osteoblasts, and a selection of the most commonly reported miRNAs involved have been summarized in Table 16.2. Some of the microRNAs studied are downregulated during normal osteogenesis, and as a result are considered as osteogenic inhibitors (such as miR-138, -141, -338), while others are upregulated and are considered osteogenic promoters (such as miR29b, -196a, 378). A selection of the miRNAs reported in literature show clear contradictory behavior (such as miR-26a, -29a, -335) with many researchers reporting either inhibitory or promoting behavior depending on their experimental conditions, which is testimony to the inherent complexity and cross-talk of the molecular mechanisms at play during the osteogenic differentiation of MSCs. Recently, many TFs have been identified to be involved in bone regeneration, however, two of the most prominent TFs in osteoblastogenesis specifically are RUNX2 and Osx (which functions downstream from RUNX2 [224]). RUNX2 functions by binding to the promotor region of osteoblast specific genes (ALP, COL1, OPN, OCN, and bone sialoprotein) and initiates transcription [225]. Furthermore, the expression of RUNX2 is significantly increased during the early stages of osteoblast differentiation at the preosteoblast stage. RUNX2 is therefore identified as an essential transcriptional marker for the detection of osteoblast differentiation. It has been shown that upregulation of RUNX2 by MSCs induces osteogenic differentiation, whilst suppression of RUNX2 induces the MSCs to undergo chondrogenic or adipogenic differentiation [226]. Although RUNX2 is essential during the preosteoblast stage, expression

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of RUNX2 has been shown to not be necessary for the continued expression of the major osteogenic genes in mature osteoblasts [227]. Many miRNA are reported to affect the expression of RUNX2, either by direct targeting, or indirectly by targeting RUNX2 inhibitors/promotors. miR-23, miR-24, miR-106a, miR-133, miR-222, miR335, miR-338 and miR-433 among others inhibit osteogenic differentiation by the direct targeting of RUNX2 [215,216,223,228]. Conversely miR-764 promotes osteoblast differentiation by inhibiting RUNX2 degradation and miR-3960 promotes the osteoblast differentiation via targeting homeobox A2 (HOXA2), a known RUNX2 inhibitor [223]. Osx functions downstream from RUNX2 [224], and is a vital transcription factor involved in osteogenesis involved in may signaling pathways, including MAPK and protein kinase D [229]. Osx functions during MSC differentiation from preosteoblasts, and for mediation of function during the mature osteoblast phase. Furthermore, BMP-2 mediated induction of Osx was inhibited by the blocking of RUNX2, further indicating the regulatory role of RUNX2 in the expression of Osx [224]. Osx-null mice die at birth due to lack of mineralized skeletons [230] and Osx-deactivated mice have been reported to experience spinal deformities [231] indicating the important role of Osx in skeletal development. miR-637 has been reported to directly target Osx, and maintains the balance between adipocytes and osteoblasts. Expression of miR-637 during osteogenic differentiation is decreased, with miR-637 supressing COL4A1 and Osx, consequently inhibiting osteogenic differentiation [221]. Furthermore, Osx has been shown to be important in osteoblast mineralization, and overexpression of Osx-targeting miR-93 has been reported to inhibit mineralization [232]. Goettsch et al. [233] reported a decrease in Osxtargeting miR-125b during the transdifferentiation of vascular smooth muscle cells, and subsequently that the inhibition of endogenous miR-125b can in fact promote osteogenesis. In addition, miR-31, miR-142 and miR-138 have been identified as Osx inhibitors [234], and miR-322 was shown to be a positive modulator of Osx, by targeting Tob2, which regulates Osx degradation [235]. There are numerous other important TFs involved in osteogenesis that are targeted and finely tuned by miRNAs, and interestingly, many of which are known to be associated to RUNX2 as downstream, upstream, cofactors or binding partners with RUNX2 [234]. Furthermore, miRNAs also participate in the mediation of the osteogenic signaling pathways such as BMP/TGF-β, Wnt/β-catenin and MAPK pathways [236]. With the vast number of miRNAs identified (with more constantly being discovered), and with the numerous targets of each miRNA, the precise effect a particular miRNA has on the osteogenic differentiation of MSCs is not well known. Therefore, miRNAs are of great interest, and worthy of much more investigation for use in enhancing bone regeneration.

16.3.3  Bone repair and miRNAs Due to the infancy of this therapeutic alternative, there exists only a few reports of miRNAs successfully utilized in vivo for the repair of nonunion. However, in patients with fractures, miR-92a is downregulated and after 21 days the levels of

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miR-92a expression are seen to recover, indicating the inhibitory effect of miR-92a on bone regeneration. To this end, Murata et al. [237] performed systematic delivery of anti-miR-26a on young mice with femoral fractures and saw an enhanced effect in fracture healing with increased callus formation and vascularization. Most often, miRNAs are utilized in a scaffold based system and have been exhibiting promising results. Downregulation of miR-31 in BMSCs has been shown to promote osteogenesis, and Deng et al. [238] seeded anti-miR-31 expressing BMSCs onto a polyglycerol sebacate scaffold, and reported effective bone repair in rats with a critical-size calvarial defect. In a similar study, BMSCs transfected with anti-miR-31 were seeded on β-TCP based scaffolds, and implanted into the medial orbital bone defects in a canine model, and efficient repair with increased bone mineral density and volume was observed [239]. Angiogenesis, along with osteogenesis, is also a necessary factor in the regeneration of healthy bone tissue, and miR-26a is a known factor in both processes. Li et al. [187] were able to exhibit improved bone repair with both osteogenesis and angiogenesis by the seeding of miR-26a transfected BMSCs on a hydrogel and implanted in a rat critical-size cavarial defect. The scaffolds in this study exhibited complete bone repair with improved vascularization, hinting at the importance of osteogenic/angiogenic coupling. Therefore, with the increased exposure of miRNA as an enhanced therapeutic option for the treatment of bone regeneration, the use of bone scaffolds containing miRNA therapeutics is certainly a promising and highly exciting area of tissue engineering.

16.4  Final conclusions In summary, the most recent advances and breakthroughs in bone regeneration strategies are highlighting the importance of the bone microenvironment, with strong interrogation of the “cellular cross-talk” and paracrine effects of any cell components that are included in such technologies. Many promising results are being reported through the cellular delivery of nucleic acids for the purposes of genetic alteration. The use of plasmid DNAs, mRNAs, siRNAs, and most recently miRNAs to introduce the upregulation of osteogenic-promoting genes or the downregulation of osteogenic-inhibiting genes in MSCs or osteoprogenitor cells has led to the creation of third-generation bone regeneration strategies that are proving to be significantly more efficacious than their second generation (biomaterial only) counterparts. Additionally, such strategies may also remove some of the associated risks with cell-seeded regeneration scaffolds (such as immunogenic rejection) as the genetic engineering of cells can be done via an autologous cell transplant. However, to further drive the advancement of such technology, a more robust understanding of the cellular mechanisms and signaling pathways involved in osteogenesis must be uncovered, to better inform future research. Furthermore, a well-chosen nucleic acid strategy is only theoretical and is inherently limited by the percentage of cells that can be successfully transfected with the intended genetic

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cargo. Therefore, selection of a suitable nonviral delivery vehicle for nucleic acid therapies is of the utmost importance to ensure the highest levels of efficacy, and the use of CPPs such as RALA are exhibiting some of the highest levels of transfection efficiency (higher than CaP or PEI delivery vectors) without the high levels of toxicity usually associated high transfection rates (such as that seen during lipofection). The combination effects of a well-chosen biomaterial in addition, to the use of a well understood nucleic acid therapy, to better mimic the native processes of bone regeneration on a molecular scale, may demonstrate some of the best results to-date for bone regeneration technologies.

References

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Further reading [1] Shah N, Steptoe RJ, Parekh HS. Low-generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA. J Pept Sci. 6(17):470–478. [2] Zorko M, Langel U. Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv Drug Deliv Rev 2005;57:529–45. [3] Maurer B, Stanczyk J, Jüngel A, et al. MicroRNA-29, a key regulator of collagen expression in systemic sclerosis. Arthritis Rheum 2010;62:1733–43.