Photoclick Chemistry: A Bright Idea.

At its basic conceptualization, photoclick chemistry embodies a collection of click reactions that are performed via the application of light. The emergence of this concept has had diverse impact over a broad range of chemical and biological research due to the spatiotemporal control, high selectivity, and excellent product yields afforded by the combination of light and click chemistry. While the reactions designated as "photoclick" have many important features in common, each has its own particular combination of advantages and shortcomings. A more extensive realization of the potential of this chemistry requires a broader understanding of the physical and chemical characteristics of the specific reactions. This review discusses the features of the most frequently employed photoclick reactions reported in the literature: photomediated azide-alkyne cycloadditions, other 1,3-dipolarcycloadditions, Diels-Alder and inverse electron demand Diels-Alder additions, radical alternating addition chain transfer additions, and nucleophilic additions. Applications of these reactions in a variety of chemical syntheses, materials chemistry, and biological contexts are surveyed, with particular attention paid to the respective strengths and limitations of each reaction and how that reaction benefits from its combination with light. Finally, challenges to broader employment of these reactions are discussed, along with strategies and opportunities to mitigate such obstacles.

Click Nucleic Acid-DNA Binding Behavior: Dependence on Length, Sequence, and Ionic Strength.

Click nucleic acids (CNAs) are a new, low-cost class of xeno nucleic acid (XNA) oligonucleotides synthesized by an efficient and scalable thiol-ene polymerization. In this work, a thorough characterization of oligo(thymine) CNA-oligo(adenine) DNA ((dA)20) hybridization was performed to guide the future implementation of CNAs in applications that rely on sequence-specific interactions. Microscale thermophoresis provided a convenient platform to rapidly and systematically investigate the effects of several factors (i.e., sequence, length, and salt concentration) on the CNA-DNA dissociation constant (Kapp). Because CNAs have limited water solubility, all studies were performed in aqueous-DMSO mixtures. CNA-DNA hybrids between oligo(thymine) CNA (average length of 16 bases) and (dA)20 DNA have good stability despite the high organic content, a favorable attribute for many emerging applications of XNAs. In particular, the Kapp of CNA-DNA hybrids in 65 vol % DMSO with 10 mM sodium chloride (NaCl) was 0.74 ± 0.1 µM, whereas the Kapp for (dT)20-(dA)20 DNA-DNA was found to be 45 ± 2 µM in a buffer without DMSO but at the same NaCl concentration. CNA hybridized with DNA following Watson-Crick base pairing with excellent sequence specificity, discriminating even a single-base-pair mismatch, with Kapp values of 0.74 ± 0.1 and 3.7 ± 0.6 µM for complementary and single-base-pair mismatch sequences, respectively. As with dsDNA, increasing CNA length led to more stable hybrids as a result of increased base pairing, where Kapp decreased from 5.6 ± 0.8 to 0.27 ± 0.1 µM as the CNA average length increased from 7 to 21 bases. However, unlike DNA-DNA duplexes, which are largely unstable at low salt concentrations, the CNA-DNA stability does not depend on salt concentration, with Kapp remaining consistent between 1.0 and1.9 µM over a NaCl concentration range of 1.25-30 mM.

Sequence-Controlled Synthesis of Advanced Clickable Synthetic Oligonucleotides.

Through thiol-ene photopolymerization of presynthesized oligomers, advanced clickable nucleic acids (CNA-2G) are synthesized with sequence-controlled repeating units. As examples, poly(thymine-adenine) (polyTA) CNA-2G and poly(thymine-thymine-cytosine) CNA-2G are synthesized by polymerizing thiol-ene heterofunctional dimers with pendant thymine-adenine nucleobases and trimer with pendant thymine-thymine-cytosine nucleobases. Based on size exclusion chromatography (SEC) analysis, polyTA and polyTTC have number average molecular weights of 2000 and 1800, respectively, which contain 7-8 pendant nucleobases. Based on the different behavior of the CNA-2G monomers and CNA-2G oligomers with two or more pendant nucleobases in photopolymerization, an unusual thiol-ene chain-growth propagation mechanism is observed for the former and a common thiol-ene step-growth propagation mechanism for the latter. The uncommon thiol-ene chain-growth propagation is hypothesized to rely on a six-membered ring mediated intramolecular hydrogen atom transfer process.

Click Nucleic Acid Mediated Loading of Prodrug Activating Enzymes in PEG-PLGA Nanoparticles for Combination Chemotherapy.

The simultaneous delivery of multiple therapeutics to a single site has shown promise for cancer targeting and treatment. However, because of the inherent differences in charge and size between drugs and biomolecules, new approaches are required for colocalization of unlike components in one delivery vehicle. In this work, we demonstrate that triblock copolymers containing click nucleic acids (CNAs) can be used to simultaneously load a prodrug enzyme (cytosine deaminase, CodA) and a chemotherapy drug (doxorubicin, DOX) in a single polymer nanoparticle. CNAs are synthetic analogs of DNA comprised of a thiolene backbone and nucleotide bases that can hybridize to complementary strands of DNA. In this study, CodA was appended with complementary DNA sequences and fluorescent dyes to allow its encapsulation in PEG-CNA-PLGA nanoparticles. The DNA-modified CodA was found to retain its enzyme activity for converting prodrug 5-fluorocytosine (5-FC) to active 5-fluorouracil (5-FU) using a modified fluorescent assay. The DNA-conjugated CodA was then loaded into the PEG-CNA-PLGA nanoparticles and tested for cell cytotoxicity in the presence of the 5-FC prodrug. To study the effect of coloading DOX and CodA within a single nanoparticle, cell toxicity assays were run to compare dually loaded nanoparticles with nanoparticles loaded only with either DOX or CodA. We show that the highest level of cell death occurred when both DOX and CodA were simultaneously entrapped and delivered to cells in the presence of 5-FC.

Encoding Stem-Cell-Secreted Extracellular Matrix Protein Capture in Two and Three Dimensions Using Protein Binding Peptides.

Capturing cell-secreted extracellular matrix (ECM) proteins through cooperative binding with high specificity and affinity is an important function of native tissue matrices during both tissue homeostasis and repair. However, while synthetic hydrogels, such as those based on poly(ethylene glycol) (PEG), are often proposed as ideal materials to deliver human mesenchymal stem cells (hMSCs) to sites of injury to enable tissue repair, they do not have this capability-a capability that would enable cells to actively remodel their local extracellular microenvironment and potentially provide the required feedback control for more effective tissue genesis. In this work, we detail a methodology that engenders poly(ethylene glycol) (PEG)-based two-dimensional substrates and three-dimensional porous hydrogels with the ability to capture desired extracellular matrix (ECM) proteins with high specificity. This "encoded" ECM protein capture is achieved by decorating the PEG-based materials with protein binding peptides (PBPs) synthesized to be specific in their binding of fibronectin, laminin, and collagen I, which are not only the most omnipresent ECM proteins in human tissues but, as we confirmed, are also secreted to differing extents by hMSCs under in vitro maintenance conditions. By encapsulating hMSCs into these PBP-functionalized hydrogels, and culturing them in protein-free maintenance media, we demonstrate that these PBPs not only actively recruit targeted ECM proteins as they are secreted from hMSCs but also retain them to much higher levels compared to nonfunctionalized gels. This novel approach thus enables the fabrication of encoded surfaces and hydrogels that capture cell-secreted proteins, with high specificity and affinity, in a programmable manner, ready for applications in many bioengineering applications, including bioactive surface coatings, bioassays, stem cell culture, tissue engineering, and regenerative medicine.

New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA.

Due to the ability to generate oligomers of precise sequence, sequential and stepwise solid-phase synthesis has been the dominant method of producing DNA and other oligonucleotide analogues. The requirement for a solid support, however, and the physical restrictions of limited surface area thereon significantly diminish the efficiency and scalability of these syntheses, thus, negatively affecting the practical applications of synthetic polynucleotides and other similarly created molecules. By employing the robust photoinitiated thiol-ene click reaction, we developed a new generation of clickable nucleic acids (CNAs) with a polythioether backbone containing repeat units of six atoms, matching the spacing of the phosphodiester backbone of natural DNA. A simple, inexpensive, and scalable route was utilized to produce CNA monomers in gram-scale, which indicates the potential to dramatically lower the cost of these DNA mimics and thereby expand the scope of these materials. The efficiency of this approach was demonstrated by the completion of CNA polymerization in 30 seconds, as characterized by size-exclusive chromatography (SEC) and infrared (IR) spectroscopy. CNA/DNA hybridization was demonstrated by gel electrophoresis and used in CdS nanoparticle assembly.

A supramolecular hydrogel prepared from a thymine-containing artificial nucleolipid: study of assembly and lyotropic mesophases.

An artificial nucleolipid containing thymine, a triazole-ring, and phosphatidylcholine (TTPC) moieties was prepared by copper catalyzed azide alkyne cycloaddition (CuAAC) under aqueous conditions. The resulting TTPC molecules assembled in situ into a fibrous aggregation. The study of the TTPC fiber assembly using XRD and NMR spectroscopy revealed that the formation of fibers was driven by the unique combination of the lipid and nucleobase moieties in the structure of TTPC. At a critical TTPC concentration, entanglement of the fibers resulted in the formation of a supramolecular hydrogel. Investigation of the lyotropic mesophases in the TTPC supramolecular hydrogel showed the presence of multiple phases including two liquid crystal phases (i.e., nematic and lamellar), which have a certain degree of structural order and are promising templates for constructing functional biomaterials.

Amine Induced Retardation of the Radical-Mediated Thiol-Ene Reaction via the Formation of Metastable Disulfide Radical Anions.

The effect of amines on the kinetics and efficacy of radical-mediated thiol-ene coupling (TEC) reactions was investigated. By varying the thiol reactant and amine additive, it was shown that amines retard thiyl radical-mediated reactions when the amine is adequately basic enough to deprotonate the thiol affording the thiolate anion, e.g., when the weakly basic amine tetramethylethylenediamine was incorporated in the TEC reaction between butyl 2-mercaptoacetate and an allyl ether at 5 mol %, the final conversion was reduced from quantitative to <40%. Alternatively, no effect is observed when the less acidic thiol butyl 3-mercaptopropionate is employed. The thiolate anion was established as the retarding species through the introduction of ammonium and thiolate salt additives into TEC formulations. The formation of a two-sulfur three-electron bonded disulfide radical anion (DRA) species by the reaction of a thiyl radical with a thiolate anion was determined as the cause for the reduction in catalytic radicals and the TEC rate. Thermodynamic and kinetic trends in DRA formations were computed using density functional theory and by modeling the reaction as an associative electron transfer process. These trends correlate well with the experimental retardation trends of various thiolate anions in TEC reactions.

Inhibition of protein and cell attachment on materials generated from N-(2-hydroxypropyl) acrylamide.

Effective control over biointerfacial interactions is essential for a broad range of biomedical applications. At this point in time, only a relatively small range of radically polymerizable monomers have been described that are able to generate low fouling polymer materials and surfaces. The most important examples that have been successfully used in the context of the reduction of nonspecific protein adsorption and subsequent cell attachment include PEG-based monomers such as poly(ethylene glycol) methacrylate (PEGMA), zwitterionic monomers such as 2-methacryloyloxyethyl phosphorylcholine and noncharged monomers such as acrylamide and N-(2-hydroxypropyl) methacrylamide (HPMAm). However, issues such as oxidative degradation and poor polymerization characteristics limit the applicability of most of these candidates. Here we have synthesized the monomer N-(2-hydroxypropyl) acrylamide (HPAm), examined its polymerization kinetics and evaluated its suitability for RAFT mediated polymerization in comparison to HPMAm. We also synthesized hydrogels using HPMAm and HPAm and evaluated the ability of HPAm polymers to occlude protein adsorption and cell attachment. In RAFT-controlled polymerization, much faster (8×) polymerization was observed for HPAm relative to HPMAm and better control was achieved over the molecular weight distribution. The performance of hydrogels prepared from HPAm in the prevention of protein adsorption and cellular attachment was equivalent to or better than that observed for materials made from HPMAm and PEG. These results open the door for HPAm based polymers in applications where effective control over biointerfacial interactions is required.

Radical-Mediated Degradation of Thiol-Maleimide Hydrogels.

Michael addition between thiol- and maleimide-functionalized molecules is a long-standing approach used for bioconjugation, hydrogel crosslinking, and the functionalization of other advanced materials. While the simplicity of this chemistry enables facile synthesis of hydrogels, network degradation is also desirable in many instances. Here, the susceptibility of thiol-maleimide bonds to radical-mediated degradation is reported. Irreversible degradation in crosslinked materials is demonstrated using photoinitiated and chemically initiated radicals in hydrogels and linear polymers. The extent of degradation is shown to be dependent on initiator concentration. Using a model linear polymer system, the radical-mediated mechanism of degradation is elucidated, in which the thiosuccinimide crosslink is converted to a succinimide and a new thioether formed with an initiator fragment. Using laser stereolithography, high-fidelity spatiotemporal control over degradation in crosslinked gels is demonstrated. Ultimately, this work establishes a platform for controllable, radical-mediated degradation in thiol-maleimide hydrogels, further expanding their versatility as functional materials.

Reversible Intracellular Gelation of MCF10A Cells Enables Programmable Control Over 3D Spheroid Growth.

In nature, some organisms survive extreme environments by inducing a biostatic state wherein cellular contents are effectively vitrified. Recently, a synthetic biostatic state in mammalian cells is achieved via intracellular network formation using bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers. In this work, the effects of intracellular network formation on a 3D epithelial MCF10A spheroid model are explored. Macromer-transfected cells are encapsulated in Matrigel, and spheroid area is reduced by ≈50% compared to controls. The intracellular hydrogel network increases the quiescent cell population, as indicated by increased p21 expression. Additionally, bioenergetics (ATP/ADP ratio) and functional metabolic rates are reduced. To enable reversibility of the biostasis effect, a photosensitive nitrobenzyl-containing macromer is incorporated into the PEG network, allowing for light-induced degradation. Following light exposure, cell state, and proliferation return to control levels, while SPAAC-treated spheroids without light exposure (i.e., containing intact intracellular networks) remain smaller and less proliferative through this same period. These results demonstrate that photodegradable intracellular hydrogels can induce a reversible slow-growing state in 3D spheroid culture.

Photoinduced Dithiolane Crosslinking for Multiresponsive Dynamic Hydrogels.

While many hydrogels are elastic networks crosslinked by covalent bonds, viscoelastic hydrogels with adaptable crosslinks are increasingly being developed to better recapitulate time and position-dependent processes found in many tissues. In this work, 1,2-dithiolanes are presented as dynamic covalent photocrosslinkers of hydrogels, resulting in disulfide bonds throughout the hydrogel that respond to multiple stimuli. Using lipoic acid as a model dithiolane, disulfide crosslinks are formed under physiological conditions, enabling cell encapsulation via an initiator-free light-induced dithiolane ring-opening photopolymerization. The resulting hydrogels allow for multiple photoinduced dynamic responses including stress relaxation, stiffening, softening, and network functionalization using a single chemistry, which can be supplemented by permanent reaction with alkenes to further control network properties and connectivity using irreversible thioether crosslinks. Moreover, complementary photochemical approaches are used to achieve rapid and complete sample degradation via radical scission and post-gelation network stiffening when irradiated in the presence of reactive gel precursor. The results herein demonstrate the versatility of this material chemistry to study and direct 2D and 3D cell-material interactions. This work highlights dithiolane-based hydrogel photocrosslinking as a robust method for generating adaptable hydrogels with a range of biologically relevant mechanical and chemical properties that are varied on demand.

Intracellular Crowding by Bio-Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis.

To survive extreme conditions, certain animals enter a reversible protective stasis through vitrification of the cytosol by polymeric molecules such as proteins and polysaccharides. In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine-mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio-orthogonal strain-promoted azide-alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5-fold. Real-time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 h vs 19 h) and decreases in motility (30 µm h-1  vs 15 µm h-1 ). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho-Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function.

Light-Activated Stress Relaxation, Toughness Improvement, and Photoinduced Reversal of Physical Aging in Glassy Polymer Networks.

A covalent adaptable network (CAN) with high glass transition temperature (Tg ), superior mechanical properties including toughness and ductility, and unprecedented spatio-temporally controlled dynamic behavior is prepared by introducing dynamic moieties capable of reversible addition fragmentation chain transfer (RAFT) into photoinitiated copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)-based networks. While the CuAAC polymerization yields glassy polymers composed of rigid triazole linkages with enhanced toughness, the RAFT moieties undergo bond exchange leading to stress relaxation upon light exposure. This unprecedented level of stress relaxation in the glassy state leads to numerous desirable attributes including glassy state photoinduced plasticity, toughness improvement during large deformation, and even photoinduced reversal of the effects of physical aging resulting in the rejuvenation of mechanical and thermodynamic properties in physically aged RAFT-CuAAC networks that undergo bond exchange in the glassy state. Surprisingly, when an allyl-sulfide-containing azide monomer (AS-N3 ) is used to form the network, the network exhibits up to 80% stress relaxation in the glassy state (Tg  - 45 °C) under fixed displacement. In situ activation of RAFT during mechanical loading results in a 50% improvement in elongation to break and 40% improvement in the toughness when compared to the same network without light-activation of RAFT during the tensile testing.

Toward Stimuli-Responsive Dynamic Thermosets through Continuous Development and Improvements in Covalent Adaptable Networks (CANs).

Covalent adaptable networks (CANs), unlike typical thermosets or other covalently crosslinked networks, possess a unique, often dormant ability to activate one or more forms of stimuli-responsive, dynamic covalent chemistries as a means to transition their behavior from that of a viscoelastic solid to a material with fluid-like plastic flow. Upon application of a stimulus, such as light or other irradiation, temperature, or even a distinct chemical signal, the CAN responds by transforming to a state of temporal plasticity through activation of either reversible addition or reversible bond exchange, either of which allows the material to essentially re-equilibrate to an altered set of conditions that are distinct from those in which the original covalently crosslinked network is formed, often simultaneously enabling a new and distinct shape, function, and characteristics. As such, CANs span the divide between thermosets and thermoplastics, thus offering unprecedented possibilities for innovation in polymer and materials science. Without attempting to comprehensively review the literature, recent developments in CANs are discussed here with an emphasis on the most effective dynamic chemistries that render these materials to be stimuli responsive, enabling features that make CANs more broadly applicable.

Flocculation behavior and mechanisms of block copolymer architectures on silica microparticle and Chlorella vulgaris systems.

HYPOTHESIS: Flocculation performance using polyelectrolytes is influenced by critical design parameters including molecular weight, amount and sign of the ionic charge, and polymer architecture. It is expected that systematic variation of these characteristics will impact not only flocculation efficiency (FE) achieved but that charge density and architecture, specifically, can alter the flocculation mechanism. Therefore, it should be possible to tune these design parameters for a desired flocculation application. EXPERIMENTS: Cationic-neutral and polyampholytic copolymers, exhibiting a range of molecular weights (103-106 g/mol), varying charge levels (0-100% cationic, neutral and anionic), and random or block copolymer architecture, were applied to dilute suspensions of silica microparticles (control) and Chlorella vulgaris. FE and zeta potential values were determined over a range of flocculant doses to evaluate effectiveness and mechanism achieved. FINDINGS: These different classes of copolymers provide specific benefits for flocculation, with many achieving >95% flocculation. Block copolymer flocculants exhibit a proposed, dominant bridging mechanism, therefore reducing flocculant dosage required for effective flocculation when compared to analogous random copolymer flocculants. Polyampholytic copolymers applied to C. vulgaris generally exhibited a bridging mechanism and increased FE compared to equivalent cationic-neutral copolymers, indicating a benefit of the anionic component on a more, complex, diversely charged suspension.

A Versatile Synthetic Extracellular Matrix Mimic via Thiol-Norbornene Photopolymerization.

Step-growth, radically mediated thiol-norbornene photopolymerization is used to create versatile, stimuli-responsive poly(ethylene glycol)-co-peptide hydrogels The reaction is cytocompatible and allows for the encapsulation of human mesenchymal stem cells with a viability greater than 95%. Cellular spreading is dictated via three-dimensional biochemical photopatterning.

Enzyme-mediated redox initiation for hydrogel generation and cellular encapsulation.

A rapid, water-soluble enzyme-mediated radical chain initiation system involving glucose oxidase and Fe(2+) generated hydrogels within minutes at 25 degrees C and in ambient oxygen. The initiation components were evaluated for their effect on polymerization rates of hydroxyethyl acrylate-poly(ethylene glycol)(575) diacrylate comonomer solutions using near-infrared spectroscopy. Increasing glucose concentration increased polymerization rates until reaching a rate plateau above 1 x 10(-3) M of glucose. A square root dependence of the initial polymerization rate on Fe(2+) concentration was observed between 1.0 x 10(-4) M and 5.0 x 10(-4) M of Fe(2+), whereupon excess Fe(2+) reduced final acrylate conversions. The glucose oxidase-mediated initiation system was employed for encapsulation of fibroblasts (NIH3T3s) into a poly(ethylene glycol) tetra-acrylate (M(n) approximately 20000) hydrogel scaffold demonstrating 96% (+/-3%) viability at 24 h postencapsulation. This first use of enzyme-mediated redox radical chain initiation for cellular encapsulation demonstrates polymerization of hydrogels in situ with kinetic control, minimal oxygen inhibition issues, and utilization of low initiator concentrations.

Phosphate-Based Cross-Linked Polymers from Iodo-ene Photopolymerization: Tuning Surface Wettability through Thiol-ene Chemistry.

Motivated by the various reported potential applications of poly(phosphine oxide) materials, a visible light photoinitiated iodo-ene reaction was successfully employed in network polymerization between the phosphorus-containing multifunctional monomer, tris(allyloxymethyl)phosphine oxide (TAOPO), and diiodoperfluorobutane. The cross-linked poly(phosphine oxide) network exhibited a higher glass transition temperature than a similarly cross-linked polymer formulated with trimethylolpropane triallyl ether (TMPTAE). Interestingly, the TMPTAE/DIPFB cross-linked polymer, changed color from clear to yellow within 10 min of exposure to air, whereas the cross-linked poly(phosphine oxide) underwent a similar change only upon heating. Upon investigation, it was determined that alkenes were generated within the polymer network, presumably via elimination, accounting for the observed color. These double bonds, formed in the polymer matrix, permitted surface modification via radical thiol-ene reaction. The successful surface functionalization with PEG-SH resulted in increasing the surface wettability. Additionally, the phosphorus-containing network polymer with double bonds in the polymer matrix showed shape memory capability, this representing an exciting and versatile materials platform.

Synthesis and Assembly of Click-Nucleic-Acid-Containing PEG-PLGA Nanoparticles for DNA Delivery.

Co-delivery of both chemotherapy drugs and siRNA from a single delivery vehicle can have a significant impact on cancer therapy due to the potential for overcoming issues such as drug resistance. However, the inherent chemical differences between charged nucleic acids and hydrophobic drugs have hindered entrapment of both components within a single carrier. While poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG-PLGA) copolymers have been used successfully for targeted delivery of chemotherapy drugs, loading of DNA or RNA has been poor. It is demonstrated that significant amounts of DNA can be encapsulated within PLGA-containing nanoparticles through the use of a new synthetic DNA analog, click nucleic acids (CNAs). First, triblock copolymers of PEG-CNA-PLGA are synthesized and then formulated into polymer nanoparticles from oil-in-water emulsions. The CNA-containing particles show high encapsulation of DNA complementary to the CNA sequence, whereas PEG-PLGA alone shows minimal DNA loading, and non-complementary DNA strands do not get encapsulated within the PEG-CNA-PLGA nanoparticles. Furthermore, the dye pyrene can be successfully co-loaded with DNA and lastly, a complex, larger DNA sequence that contains an overhang complementary to the CNA can also be encapsulated, demonstrating the potential utility of the CNA-containing particles as carriers for chemotherapy agents and gene silencers.