DNA-based drug carriers and dynamic proteoids with tunable properties Liu, Yun
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Chapter 1
Nucleic Acid Amphiphiles as Drug Carriers
This chapter gives an introduction on the development of nucleic acid amphiphiles as drug carriers, including their synthetic approaches, controllable self-assembly behavior, and modification methods.
1.1 Introduction
Drug discovery is a challenging and time-consuming undertaking. Over the past decades, new technologies in drug discovery have emerged, including combinatorial chemistry,[1] structure-based drug design,[2] high-throughput screening,[3] fragment-based drug design[4] and ultrahigh-throughput screening.[5] With these methodologies, numerous pharmacologically interesting molecules have been created and identified. Increasingly, people focus on optimizing their properties from already in the early stage of drug development. Despite this, it is surprising that more than 40% of approved drugs on the market and about 90% of drug candidates in the discovery pipeline are poorly soluble in water.[6]
Poor water solubility is undesirable as it slows down the absorption process, thereby leading to inadequate and variable bioavailability. Increasing the dosage is necessary to achieve the required concentration in the systemic circulation at which the desired therapeutic response may take place.[7] In most cases, this causes toxicity issues and adverse effects. Therefore, research efforts have been focused on establishing drug delivery approaches and numerous delivery techniques have been reported and developed (Figure 1).[8]
Among all approaches currently available, nanomedicine has emerged as a particularly active field of research. Nanomedicine is the application of nanotechnology to medicine.[9] One of its applications is to utilize nanoparticles as drug-delivery vehicles. These nanocarriers generally range from 1 to 1000 nm in diameter. Typically, the drug is dissolved, entrapped, adsorbed, attached or encapsulated into or onto nanocarriers. Based on their material composition, nanocarriers are divided into three types (Figure 2): bio-inspired nanocarriers,[10] inorganic nanocarriers[11] and synthetic polymeric nanocarriers.[12] The increasing research interest in synthetic polymers for drug delivery is due to their diversity, which is attributed to the diversity of synthetic polymers. It endows the resulting nanocarriers with multiple capacities, such as stimuli-responsiveness (pH,[13] temperature,[14] light,[15] etc.), bioavailabity,[16] and self-assembly.[17] According to their structural features, synthetic polymeric nanocarriers can be classified into dendrimers,[18] polymer–drug conjugates[19] and self-assembled nanocarriers.[20]
Figure 2. Categories of nanocarriers.
Molecular self-assembly is omnipresent in nature and plays an essential role during the formation of many biological structures.[21] Furthermore, it is an important approach to create well-defined and controllable nanostructures, a concept referred to as bottom-up construction.[22] Molecular self-assembly allows the fabrication of
different types of organized nanostructures from simple building blocks. The whole process is driven by non-covalent interactions, such as hydrogen bonding, electrostatic, van der Waals and hydrophobic interactions, does not require any external input and depends on the inherent nature of molecules themselves. Numerous types of molecules and materials have been utilized to fabricate self-assembled nanocarriers, resulting in nanocarriers with diverse nanostructures and properties for various medical conditions and drugs. Because of their diversity and infinite potential, self-assembled nanocarriers have drawn great research attention over the past decades. Nanocarriers are formed through self-assembly of various molecules, including nanospheres,[23] nanocapsules,[24] micelles,[25] polymersomes,[26] nanogels,[27] nanofibers (Figure 3).[28] Desired structures of the resulting nanocarriers can be tuned by designing and synthesizing the building blocks at a molecular level.
1.2 Self-assembled DNA nanocarriers
DNA is a material that can self-assemble into double helices through Watson-Crick base pairing, and the structure is stabilized by non-covalent interactions, including hydrogen-bonding, π−π-stacking and hydrophobic interactions. The strict base-pairing principles enable DNA to construct various types of well-ordered nanostructures (from 1D to 3D structures) for a range of medical applications.[29]
Structural DNA nanotechnology[30] was pioneered around three decades ago by Seeman, who performed research with DNA junctions and lattices.[31] Since then, a rapid growth and development has been witnessed in this field. The key contribution to this research area is the invention of DNA origami in 2006,[32] which is the nanoscale folding of DNA used to create two- and three-dimensional nanostructures through Watson-Crick base pairing. To date, a variety of DNA nanostructures have been successfully constructed and applied as targeted drug carriers. The inherent properties of DNA endow these nanocarriers with improved biocompatibility and biodegradability. Based on the molecular composition of DNA nanocarriers, they are grouped into pristine DNA nanocarriers and hybrid DNA nanocarriers.
Structural DNA nanotechnology allows scientists to engineer pristine DNAs into nanocarriers with well-defined structures, such as cubic, tetrahedral, octahedral, buckyball, icosahedra and origami.[33] This topic has been recently reviewed and will not be covered in detail in this chapter.[34] The assembled nanocarriers have several advantages, including: (1) programmable and well-ordered structure, (2) high loading capacity, (3) good structural stability in a physiological environment. Aside from pristine DNA, DNA-polymer or -lipid hybrid macromolecules are also exploited as
building blocks to construct self-assembled DNA nanocarriers and will be discussed in section 1.3.
Figure 3. Schematic representations of various polymeric nanocarriers.
1.3 Amphiphilic DNA nanocarriers
The self-assembly of DNA-polymer or -lipid conjugates generates various nanostructures such as hydrogels, micelles,[35] vesicles and tubes.[36] Hydrogels formed from DNA hybrid materials have been reviewed elsewhere.[37] In this chapter, the focus is on DNA amphiphiles self-assembling into micelles, tubes and vesicles are focused (Figure 4), especially micelles.
Nucleic acid amphiphiles consist of a hydrophilic DNA moiety and a hydrophobic segment. They are synthesized and used as amphiphilic DNA nanocarriers by self-assembling into micelles, tubes or vesicles. In aqueous solution, these structures are stabilized by both non-covalent and hydrophobic interactions from the DNA and hydrophobic moieties, respectively. For micelles and tubes, DNAs are exposed to the aqueous environment, so they have a hydrophobic interior and a hydrophilic external corona. Therefore, hydrophobic drugs can be encapsulated inside the core. In contrast, vesicles are composed of an amphiphilic bilayer, such that both the interior and exterior are hydrophilic, with a hydrophobic layer in between. The latter is suitable for insoluble drugs, while hydrophilic drugs can be encapsulated in the hydrophilic interior. As a result, vesicles are capable of delivering both hydrophobic drugs and
hydrophilic drugs. Given that the drug interacts with the drug carrier through non-covalent interactions, there is no need for covalent modification of the drug.
Figure 4. Schematic representation of the self-assembly of DNA amphiphiles into
micelles, vesicle and tubes.
Owing to their small size and use of biocompatible DNA as a component, these nanocarriers feature advantages including: (1) high loading capacity attributed to large surface area to volume ratio; (2) improved biocompatibility by reducing the dose; (3) passive targeting to and drug accumulation within tumors because of the enhanced permeability and retention (EPR)[38] effect of the vasculature; (4) ease of manufacture; (5) compatibility with a wide range of drugs; (6) automated synthesis; (7) active targeting to specific sites and controllable activation of drugs given their responsiveness to internal or external stimuli; (8) active targeting to specific sites through ligand-receptor-mediated drug targeting.[39] Furthermore, these advantages can be of benefit to almost all drug-administration routes, namerly parenteral, oral, nasal and ocular administrations.
In some diseases, especially tumors, there is an overexpression of cell surface tumor-associated receptors on the cell surface that are found at low levels in normal tissues. In this case, by functionalizing the surface of nanocarriers with specific ligands for these receptors, an increased accumulation and selective cellular uptake could be expected. As a result, the therapeutic efficacy of the drug is improved
leading to a lower dose. The following ligands have been investigated in various drug-delivery systems: carbohydrates,[40] monoclonal antibodies,[41] folate,[42] homing peptides,[43]targeting proteins[44] and aptamers.[45]
1.3.1 Synthesis of DNA-polymer or -lipid conjugates
DNA-polymer conjugates were first synthesized in the 1980s by grafting DNA onto a poly((L-lysine) backbone.[46] Since then, various synthetic methods have been exploited to obtain DNA-polymer (including hydrophobic and hydrophilic polymers) or -lipid conjugates, using chemical synthesis and molecular biology techniques (Figure 5). It is noteworthy that these synthetic methods can also be used for the surface modification and functionalization of the nanocarriers.
Figure 5. Methods explored for chemical synthesis of DNA hybrid materials.
Through chemical synthesis, designed and specific DNA sequences can be obtained. Generally, three types of strategies are reported for chemical synthesis: (1) Solution-phase synthesis. DNA and polymer are independently synthesized through standard methods, followed by coupling them in solution. The advantage of this strategy is the ease of functionalization and diversity allowed for the polymer moiety. However, it is not an easy task to find the proper solvent for the reaction. Poor solubility of hydrophobic polymers in aqueous media generally leads to low yields. In
contrast, hydrophilic polymers can be coupled relatively easily to DNA given that both are sufficiently soluble in aqueous media. Different conjugation reactions have been used for the coupling in aqueous solution, including click chemistry,[47] Michael addition[48] and the formation of disulfide[49] and amide bonds (Figure 5a).[50] (2) Polymer-linked phosphoramidites or lipid-modified nucleobases are used for solid-phase synthesis (Figure 5b).[51] The essential step of this method is the functionalization of the polymer or the modification of the nucleobase with a polymer/lipid. As a result, the product can be directly applied in standard solid-phase synthesis of DNA. For instance, Herrmann and coworkers designed and synthesized a family of lipid-modifed DNA 12-mers by using dodec-1-yne-modified uracil. The hydrophobicity of the lipid-modified DNAs is tuned by adjusting the number and position of the modified uracils.[52] Since the functionalized nucleobases can be attached in a sequence-defined manner, the key benefit of this method is the tunability. A drawback might be the poor solubility of the functionalized polymer or modified nucleobases. (3) DNA with a polymerization initiator is synthesized through solid-phase synthesis (Figure 5c), followed by polymerization in solution after cleavage or on the solid support prior to cleavage. In this method, the issue of solubility is circumvented. The former requires the initiator to be stable in solution, while the resultant DNA-polymer conjugates need to survive the cleavage step in the
latter case. For example, Das and coworkers prepared
DNA-b-poly[oligo(ethyleneoxide) methacrylate] (POEOMA) both on solid support and in solution after cleavage of DNA. DNA-b-poly(benzyl methacrylate) (PBnMA) was also synthesized after cleavage.[53] Importantly, a phosphoramidite containing a bromoisobutyryl atom-transfer radical polymerization (ATRP) initiator capable of tolerating typical DNA deprotection conditions is necessary for solution-phase initiated polymerization.
Application of molecular-biology techniques offers another way for fabrication of DNA hybrid materials. The limitation of the length of DNA imposed by solid-phase synthesis can be circumvented, by utilizing various molecular-biology techniques, enabling the synthesis of conjugates with thousands of base pairs in the DNA moiety. Such techniques include the polymerase chain reaction (PCR)[54] and enzymatic restriction and ligation.[55]
1.3.2 Controllable self-assembly of amphiphilic DNAs
When amphiphilic DNAs are dissolved in aqueous media, self-assembly takes place to form in principle various well-organized nanostructures. In practice, mainly micelles are observed. The first formation of vesicles was reported in 2007.[56] By
controlling the self-assembly of the materials, the function will be improved and the scope of potential applications expanded. Control of self-assembly is achieved in various ways.
Figure 6. Schematic representation of hybridization of DNA-b-polypropylene oxide
(PPO) micelles with various DNA molecules. (a) Base pairing with a short complementary DNA (cDNA) yields micelles. (b) Hybridization with long cDNA templates results in nanorods consisting of two double helices aligned in parallel.[58]
The first strategy is to adjust the chemical composition of the building block. Self-assembly of amphiphilic DNAs is driven by hydrophobic interactions from the polymer moiety, and the well-ordered nanostructures are stabilized by both hydrophobic interactions and the hydrophilic layer. As a result, the chemical composition has a great influence on the resultant structures. By altering the hydrophobic to hydrophilic ratio or the position of hydrophobic moieties, properties of nanostructures formed (size, shape etc.) are tunable. As mentioned in section 1.3.1, Herrmann and coworkers synthesized 12-mer lipid-modified DNAs with different numbers and positions of lipid chains. When increasing the numbers of lipid chains from two to four, the hydrophobicity is enhanced, leading to a decrement in both size
and critical micelle concentration (CMC).[51] Besides the hydrophobic moiety, adjusting the length of the DNA segment is also used to control self-assembly.[57]
One of the key advantages of amphiphilic DNAs is their capacity of hybridization with complementary DNAs (cDNAs) even after self-assembly. This ability can be used for functionalization (see section 1.3.3) to control self-assembly. Herrmann and coworkers reported for instance that upon addition of the cDNAs, micelles were switched into nanorods (Figure 6).[58]
Other ways to control self-assembly include application of enzymatic reactions[59] and change of environmental conditions, such as pH,[61] metal ions [50]and so on.
1.3.3 Modifications of amphiphilic DNAs
Further modification can be performed on the surface of nanocarriers, which corresponds to the DNA moiety. The modification can be accomplished through chemical or physical methods. The former consists in coupling functional groups during the synthesis of amphiphilic DNA (see section 1.3.1). Physical modification is achieved by hybridization of amphiphilic DNAs with cDNAs bearing the desired functional groups. The main advantage of physical modification is the reversibility. Surface modifications are employed for two main purposes: (1) to improve the biocompatibility and reduce the immunogenicity by functionalization with polyethylene glycol (PEG).[61] This can be easily realized through hybridization with PEG-cDNA strands, while the PEG-cDNA moiety also can be replaced through hybridization by adding pristine DNAs. (2) to functionalize the nanocarriers with targeting and cell-penetrating groups, such as carbohydrates, antibodies, folate, homing peptides, targeting proteins, aptamers or cell-penetrating peptides through hybridization.[62]Herrmann and coworkers reported that DNA-b-PPO micelles were successfully functionalized with folate-modified cDNAs through hybridization. This material was used as a drug carrier of doxorubicin. Cell-culture experiments showed that cell uptake is related to the density of folate on the surface, and cancer cells were efficiently killed (Figure 7).[63]
It should be pointed out that hybridization offers a potential way for multifunctionalization of DNA nanocarriers. Since cDNAs bearing different functional groups can be used for hybridization, by adding several types of modified cDNAs at once and controlling their ratios, multifunctionalization can be accomplished simultaneously in one system (Figure 8). This method is more convenient, rational, flexible and controllable compared with other methods for surface modification of drug carriers. A possible disadvantage of this method may be that it might change the self-assembly of the resultant system. One conceivable
solution is to stabilize the micelle structures by crosslinking before hybridization. On the other hand, the multifunctionalization strategy can also be applied to multifunctionalize the surface of inorganic nanoparticles, which are rigid nanocarriers. In brief, careful design and intensive research are necessary to develop an efficient multifunctional delivery system.
Figure 7. Schematic representation of the drug delivery system based on DNA-b-PPO
micelles. (a) The micelles are functionalized through hybridization with folate (red dots) modified cDNA. (b) The anticancer drug doxorubicin (green dots) is loaded into micelles. [63]
Figure 8. Schematic representation of the drug-delivery system based on
lipid-modified DNA micelles. (a) The micelles are functionalized with polyethylene glycol (PEG) and targeting groups at once through hybridizing with modified cDNA. (b) The hydrophobic drug is loaded into the micelles.
1.4 Conclusions and outlook
In this chapter, self-assembled DNA nanocarriers especially amphiphilic DNA nanocarriers are introduced and discussed, including their synthesis, controllable self-assembly and surface functionalization. The nanocarriers obtained can be readily
decorated with functional groups to target specific sites of the body, elongate the circulation time, respond to internal or external stimuli, penetrate the cell membrane, reduce immunogenicity and incorporate drugs. Both chemical modification and hybridization with cDNAs are commonly employed for functionalization. Recent progress demonstrates the potentially wide usage of the controllable and targeted drug carriers in the field of nanomedicine.
So far, structural DNA nanotechnology, and in particular drug-delivery research, remain in their early stages. Amphiphilic DNAs have shown outstanding performance as future drug carriers and need to be extensively studied to gain a better understanding enabling applications. To the best of our knowledge, no clinical trials are in progress yet. Despite of the rapid growth and development over the past decades, there are still several key issues that need to be solved prior to any medical or clinical applications. First, like all other micellar systems, their instability upon exposure to the cellular environment remains a major concern for drug delivery. Incorporating cross-linkers may prevent disassociation and might be a solution.[64] Second, the mechanism for cell entry remains unclear. Although ready cell uptake of amphiphilic DNA nanocarriers has been observed, unraveling the underlying mechanism will help to improve the delivery efficiency. Important questions to be addressed are: how do key factors such as size and shape influence cell entry. Third, safety issues, such as their intracellular route and fate, the immune response due to the high local DNA concentration and the relationship between the intracellular behavior and their physical/chemical properties, shape and size need to be investigated. Even if DNA is more biocompatible and biodegradable than other materials, the human body is so complex that DNA may still have biological effects on the host, which have not been anticipated during the design of the system. Without enough data and careful analysis, overestimates of the functionality and misleading prediction of the clinical effects might occur. Finally, method and cost of the large-scale production have always been a major challenge for synthetic DNA nanocarriers due to low yields. The synthesis of starting materials and purification on large scale are more expensive than for other materials such as polysaccharides and poly-amino acids. As this is a constantly growing and developing research field, the production needs to be scaled up, leading to a lower cost in the future. A few efforts have been made to address these problems. For instance, the production of pure DNA origami has been scaled up to gram scale from micro-/milligram scale.[65]
Taking into consideration all the advantages and disadvantages, self-assembled DNA nanocarriers are holding a great promise to be employed in nanomedicine. We anticipate multiple in vivo applications in the near future.
1.5 Outline of this thesis
Chapter 2 gives an introduction on the development of constitutionally dynamic covalent analogues of nucleic acids (DyNAs), polysaccharides (glycodynamers) and proteins (dynamic proteoids) as novel functional biomaterials, including their synthetic approaches and various applications.
In chapter 3, we report on the design and solid-phase synthesis of three types of lipid-modified DNAs. Their self-assembled micelles were ultilized as solubilizers of the insoluble photosensitizer Foscan used in photodynamic therapy. High drug loading capacities were achieved while not influence the biological activity of the active pharmaceutical ingredient.
In chapter 4, describes the use of lipid-modified DNA that forms micelles at comparatively low critical micelle concentration to render budesonide, a hydrophobic glucocorticoid with high anti-inflammatory activity, water soluble with a high loading capacity. The inhibition of interleukin-8 release showed that the new delivery system retains the inhibitory activity in cell-based assays.
In chapter 5, describes the design and synthesis of a range of dynamic proteoids through polycondensation of different types of amino acid hydrazides with a nonbiological dialdehyde. The polymerization is driven by the self-organization/folding of the generated polymers. It demonstrates that the side chains of the amino acid hydrazides have a strong influence on the rates of polymerization, structures and dynamic properties of the resulting biodynamers.
In chapter 6, we report on the design and synthesis of dynamic proteoids through polycondensation of different types of amino acid and dipeptide hydrazides with a nonbiological aromatic dialdehyde and a biological aliphatic dialdehyde. By using the biological dialdehyde, biocompatibility of the resulting biodynamers was improved. Further study demonstrated that the aromatic group in the side chain of the amino acid plays an essential role through π−π-stacking interactions, and the hydroxyl group has a less important effect and stabilizes the architectures formed via hydrogen bonds, whereas a high density of positive charges hinders the generation of biodynamers owing to the electrostatic repulsions.
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