Adamantoid Scaffolds for Multiple Cargo Loading and Cellular Delivery as ß-Cyclodextrin Inclusion Complexes.

There is huge demand for developing guests that bind ß-CD and can conjugate multiple cargos for cellular delivery. We synthesized trioxaadamantane derivatives, which can conjugate up to three cargos per guest. 1 H NMR titration and isothermal titration calorimetry revealed these guests form 1 : 1 inclusion complexes with ß-CD with association constants in the order of 103  M-1 . Co-crystallization of ß-CD with guests yielded crystals of their 1 : 1 inclusion complexes as determined by single-crystal X-ray diffraction. In all cases, trioxaadamantane core is buried within the hydrophobic cavity of ß-CD and three hydroxyl groups are exposed outside. We established biocompatibility using representative candidate G4 and its inclusion complex with ß-CD (ß-CD⊂G4), by MTT assay using HeLa cells. We incubated HeLa cells with rhodamine-conjugated G4 and established cellular cargo delivery using confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) analysis. For functional assay, we incubated HeLa cells with ß-CD-inclusion complexes of G4-derived prodrugs G6 and G7, containing one and three units of the antitumor drug (S)-(+)-camptothecin, respectively. Cells incubated with ß-CD⊂G7 displayed the highest internalization and uniform distribution of camptothecin. ß-CD⊂G7 showed higher cytotoxicity than G7, camptothecin, G6 and ß-CD⊂G6, affirming the efficiency of adamantoid derivatives in high-density loading and cargo delivery.

DNA-π Amphiphiles: A Unique Building Block for the Crafting of DNA-Decorated Unilamellar Nanostructures.

The unparalleled ability of DNA to recognize its complementary strand through Watson and Crick base pairing is one of the most reliable molecular recognition events found in natural systems. This highly specific sequence information encoded in DNA enables it to be a versatile building block for bottom-up self-assembly. Hence, the decoration of functional nanostructures with information-rich DNA is extremely important as this allows the integration of other functional molecules onto the surface of the nanostructures through DNA hybridization in a highly predictable manner. DNA amphiphiles are a class of molecular hybrids where a short hydrophilic DNA is conjugated to a hydrophobic moiety. Since DNA amphiphiles comprise DNA as the hydrophilic segment, their self-assembly in aqueous medium always results in the formation of nanostructures with shell made of DNA. This clearly suggests that self-assembly of DNA amphiphiles is a straightforward strategy for the ultradense decoration of a nanostructure with DNA. However, initial attempts toward the design of DNA amphiphiles were primarily focused on long flexible hydrocarbon chains as the hydrophobic moiety, and it has been demonstrated in several examples that they typically self-assemble into DNA-decorated micelles (spherical or cylindrical). Hence, molecular level control over the self-assembly of DNA amphiphiles and achieving diverse morphologies was extremely challenging and unrealized until recently.In this Account, we summarize our recent efforts in the area of self-assembly of DNA amphiphiles and narrate the remarkable effect of the incorporation of a large π-surface as the hydrophobic domain in the self-assembly of DNA amphiphiles. Self-assembly of DNA amphiphiles with flexible hydrocarbon chains as the hydrophobic moiety is primarily driven by the hydrophobic effect. The morphology of such nanostructures is typically predicted based on the volume ratio of hydrophobic to hydrophilic segments. However, control over the self-assembly and prediction of the morphology become increasingly challenging when the hydrophobic moieties can interact with each other through other noncovalent interactions. In this Account, the unique self-assembly behaviors of DNA-π amphiphiles, where a large π-surface acts as the hydrophobe, are described. Due to the extremely strong π-π stacking in aqueous medium, the assembly of the amphiphile is found to preferably proceed in a lamellar fashion (bilayer) and hence the morphology of the nanostructures can easily be tuned by the structural modification of the π-surface. Design principles for crafting various DNA-decorated lamellar nanostructures including unilamellar vesicles, two-dimensional (2D) nanosheets, and helically twisted nanoribbons by selecting suitable π-surfaces are discussed. Unilamellar vesicular nanostructures were achieved by using linear oligo(phenylene ethynylene) (OPE) as the hydrophobic segment, where lamellar assembly undergoes folding to form unilamellar vesicles. The replacement of OPE with a strongly π-stacking hydrophobe such as hexabenzocoronene (HBC) or tetraphenylethylene (TPE) provides extremely strong π-stacking compared to OPE, which efficiently directed the 2D growth for the lamellar assembly and led to the formation of 2D nanosheets. A helical twist in the lamella was achieved by the replacement of HBC with hexaphenylbenzene (HPB), which is the twisted analogue of HBC, directing the assembly into helically twisted nanoribbons. The most beneficial structural feature of this kind of nanostructure is the extremely dense decoration of their surface with ssDNA, which can further be used for DNA-directed organization of other functional nanomaterials. By exploring this, their potential as a nanoscaffold for predefined assembly of plasmonic nanomaterials into various plasmonic 1D, 2D, and 3D nanostructures through DNA hybridization is discussed. Moreover, the design of pH-responsive DNA-based vesicles and their application as a nanocarrier for payload delivery is also demonstrated.

Galactose Grafted Two-Dimensional Nanosheets as a Scaffold for the In Situ Synthesis of Silver Nanoparticles: A Potential Catalyst for the Reduction of Nitroaromatics.

Two major hurdles in NP-based catalysis are the aggregation of the NPs and their recycling. Immobilization of NPs onto a 2D support is the most promising strategy to overcome these difficulties. Herein, amphiphilicity-driven self-assembly of galactose-hexaphenylbenzene-based amphiphiles into galactose-decorated 2D nanosheet is reported. The extremely dense decoration of reducing sugar on the surface of the sheets is used for the in situ synthesis and immobilization of ultrafine catalytically active AgNPs by using Tollens' reaction. The potential of the system as a catalyst for the reduction of various nitroaromatics is demonstrated. Enhanced catalytic activity is observed for the immobilized AgNPs when compared to the corresponding discrete AgNPs. Recovery of the catalytic system from the reaction mixture by ultrafiltration and its subsequent recycling for several cycles without dropping its activity is shown. This is the first report demonstrating the in situ synthesis and immobilization of ultrafine AgNPs onto a 2D nanosheet that exhibits excellent catalytic performance for the reduction of nitroaromatics.

Biotin-decorated NIR-absorbing nanosheets for targeted photodynamic cancer therapy.

Targeted photodynamic therapy (PDT) is one of the promising approaches for the selective killing of cancerous cells without affecting the normal cells, and hence designing new strategies for targeted PDT is extremely important. Herein we report the design and synthesis of a new class of nanosheets derived from the self-assembly of the iodo-BODIPY-biotin conjugate as a photosensitizer for targeted PDT applications. The nanosheet exhibits a high extinction coefficient in the NIR region, high singlet oxygen efficiency, no toxicity in the dark and cell targeting ligands (biotin) on the surface, which are necessary features required for an ideal photosensitizer. Overexpression of sodium-dependent multivitamin transporters (SMVTs) in HeLa and A549 (biotin receptor positive cell lines) is explored for the selective uptake of the nanophotosensitizer through receptor mediated endocytosis (interaction between biotin and SMVT). Control experiments using a biotin receptor negative cell line (WI-38) are also carried out to confirm that the specific interaction between the SMVTs and biotin is mainly responsible for the selective uptake of the photosensitizer. Efficient killing of cancerous cells is demonstrated upon light irradiation through the generation of singlet oxygen and other reactive oxygen species around the cellular environment.

Galactose-Grafted 2D Nanosheets from the Self-Assembly of Amphiphilic Janus Dendrimers for the Capture and Agglutination of Escherichia coli.

High aspect ratio, sugar-decorated 2D nanosheets are ideal candidates for the capture and agglutination of bacteria. Herein, the design and synthesis of two carbohydrate-based Janus amphiphiles that spontaneously self-assemble into high aspect ratio 2D sheets are reported. The unique structural features of the sheets include the extremely high aspect ratio and dense display of galactose on the surface. These structural characteristics allow the sheet to act as a supramolecular 2D platform for the capture and agglutination of E. coli through specific multivalent noncovalent interactions, which significantly reduces the mobility of the bacteria and leads to the inhibition of their proliferation. Our results suggest that the design strategy demonstrated here can be applied as a general approach for the crafting of biomolecule-decorated 2D nanosheets, which can perform as 2D platforms for their interaction with specific targets.

DNA-Decorated, Helically Twisted Nanoribbons: A Scaffold for the Fabrication of One-Dimensional, Chiral, Plasmonic Nanostructures.

Crafting of chiral plasmonic nanostructures is extremely important and challenging. DNA-directed organization of nanoparticle on a chiral template is the most appealing strategy for this purpose. Herein, we report a supramolecular approach for the design of DNA-decorated, helically twisted nanoribbons through the amphiphilicity-driven self-assembly of a new class of amphiphiles derived from DNA and hexaphenylbenzene (HPB). The ribbons are self-assembled in a lamellar fashion through the hydrophobic interactions of HPB. The transfer of molecular chirality of ssDNA into the HPB core results in the bias of one of the chiral propeller conformations for HPB and induces a helical twist into the lamellar packing, and leads to the formation of DNA-wrapped nanoribbons with M-helicity. The potential of the ribbon to act as a reversible template for the 1D chiral organization of plasmonic nanomaterials through DNA hybridization is demonstrated.

DNA-Decorated Two-Dimensional Crystalline Nanosheets.

Design and synthesis of high aspect ratio 2D nanosheets with surface having ultradense array of information-rich molecule such as DNA is extremely challenging. Herein, we report a universal strategy based on amphiphilicity-driven self-assembly for the crafting of high aspect ratio, 2D sheets that are densely surface-decorated with DNA. Microscopy and X-ray analyses have shown that the sheets are crystalline. The most unique feature of the sheets is DNA-directed surface addressability, which is demonstrated through the decoration of either faces of the sheet with gold nanoparticles through sequence-specific DNA hybridization. Our results suggest that this design strategy can be applied as a general approach for the synthesis of DNA decorated high aspect ratio sheets, which may find potential applications in materials science, drug delivery, and nanoelectronics.

A pH-Responsive DNAsome from the Self-Assembly of DNA-Phenyleneethynylene Hybrid Amphiphile.

A pH-responsive DNAsome derived from the amphiphilicity-driven self-assembly of DNA amphiphile containing C-rich DNA sequence is reported. The acidification of DNAsome induces a structural change of C-rich DNA from random coil to an i-motif structure that triggers the disassembly of DNAsome and its subsequent morphological transformation into an open entangled network. The encapsulation of a hydrophobic guest into the membrane of DNAsome and its pH-triggered release upon acidification of DNAsome is also demonstrated.

Synthesis and self-assembly of DNA-chromophore hybrid amphiphiles.

DNA based spherical nanostructures are one of the promising nanostructures for several biomedical and biotechnological applications due to their excellent biocompatibility and DNA-directed surface addressability. Herein, we report the synthesis and amphiphilicity-driven self-assembly of two classes of DNA (hydrophilic)-chromophore (hydrophobic) hybrid amphiphiles into spherical nanostructures. A solid-phase "click" chemistry based modular approach is demonstrated for the synthesis of DNA-chromophore amphiphiles. Various spectroscopic and microscopic analyses reveal the self-assembly of the amphiphiles into vesicular and micellar assemblies with the corona made of hydrophilic DNA and the hydrophobic chromophoric unit as the core of the spherical nanostructures.

DNA-Based Oligochromophores as Light-Harvesting Systems.

The chromophores ethynyl pyrene as blue, ethynyl perylene as green and ethynyl Nile red as red emitter were conjugated to the 5-position of 2'-deoxyuridine via an acetylene bridge. Using phosphoramidite chemistry on solid phase labelled DNA duplexes were prepared that bear single chromophore modifications, and binary and ternary combinations of these chromophore modifications. The steady-state and time-resolved fluorescence spectra of all three chromophores were studied in these modified DNA duplexes. An energy-transfer cascade occurs from ethynyl pyrene over ethynyl perylene to ethynyl Nile red and subsequently an electron-transfer cascade in the opposite direction (from ethynyl Nile red to ethynyl perylene or ethynyl pyrene, but not from ethynyl perylene to ethynyl pyrene). The electron-transfer processes finally provide charge separation. The efficiencies by these energy and electron-transfer processes can be tuned by the distances between the chromophores and the sequences. Most importantly, excitation at any wavelength between 350 and 700 nm finally leads to charge separated states which make these DNA samples promising candidates for light-harvesting systems.

Self-assembly of DNA-oligo(p-phenylene-ethynylene) hybrid amphiphiles into surface-engineered vesicles with enhanced emission.

Surface-addressable nanostructures of linearly π-conjugated molecules play a crucial role in the emerging field of nanoelectronics. Herein, by using DNA as the hydrophilic segment, we demonstrate a solid-phase "click" chemistry approach for the synthesis of a series of DNA-chromophore hybrid amphiphiles and report their reversible self-assembly into surface-engineered vesicles with enhanced emission. DNA-directed surface addressability of the vesicles was demonstrated through the integration of gold nanoparticles onto the surface of the vesicles by sequence-specific DNA hybridization. This system could be converted to a supramolecular light-harvesting antenna by integrating suitable FRET acceptors onto the surface of the nanostructures. The general nature of the synthesis, surface addressability, and biocompatibility of the resulting nanostructures offer great promises for nanoelectronics, energy, and biomedical applications.

Bimetallic DNAsome Decorated with G4-DNA as a Nanozyme for Targeted and Enhanced Chemo/Chemodynamic Cancer Therapy.

Cancer is indisputably one of the major threats to mankind, and hence the design of new approaches for the improvement of existing therapeutic strategies is always wanted. Herein, the design of a tumor microenvironment-responsive, DNA-based chemodynamic therapy (CDT) nanoagent with dual Fenton reaction centers for targeted cancer therapy is reported. Self-assembly of DNA amphiphile containing copper complex as the hydrophobic Fenton reaction center results in the formation of CDT-active DNAsome with Cu2+-based Fenton catalytic site as the hydrophobic core and hydrophilic ssDNA protrude on the surface. DNA-based surface addressability of the DNAsome is then used for the integration of second Fenton reaction center, which is a peroxidase-mimicking DNAzyme noncovalently loaded with Hemin and Doxorubicin, via DNA hybridization to give a CDT agent having dual Fenton reaction centers. Targeted internalization of the CDT nanoagent and selective generation of •OH inside HeLa cell are also shown. Excellent therapeutic efficiency is observed for the CDT nanoagent both in vitro and in vivo, and the enhanced efficacy is attributed to the combined and synergetic action of CDT and chemotherapy.

G4-Hemin-loaded 2D nanosheets for combined and targeted chemo-photodynamic cancer therapy.

Synergetic combination therapy is emerging as one of the most promising approaches for cancer treatment. Among the various therapeutic approaches, PDT has received particular attention due to its non-invasive nature. However, the therapeutic performance of PDT is severely affected by tumour hypoxia. Herein, we report a supramolecular strategy for the fabrication of a PDT-active 2D nanosheet loaded with a POD mimicking DNAzyme for the synergetic combination of PDT and CDT for targeted cancer therapy. Assembly of biotin-functionalized BODIPY (1) and cationic ß-cyclodextrin (ß-CD+) leads to the formation of a 1/ß-CD+ nanosheet with positively charged ß-CD+ on the surface of the sheet. The cationic face of the 1/ß-CD+ sheet was then loaded with a POD-mimicking Hem-loaded G-quadruplex aptamer (Hem/DNA1) via electrostatic interactions (1/ß-CD+/Hem/DNA1). Cellular internalization of the 1/ß-CD+/Hem/DNA1 nanosheet occurs via a receptor-mediated endocytic pathway, which then undergoes lysosomal escape. Subsequently, Hem/DNA1 on the surface of 1/ß-CD+/Hem/DNA1 reacts with endogenous H2O2via the Fenton pathway to produce ˙OH and O2. Moreover, under cellular conditions, Hem inside the 1/ß-CD+/Hem/DNA1 nanosheet produces Fe2+, which then undergoes another Fenton reaction to produce ˙OH and O2. The Fe3+ generated after the Fenton reaction is then reduced in situ to Fe2+ by glutathione for the next Fenton cycle. At the same time, photoirradiation of the 1/ß-CD+ nanosheet using a 635 nm laser produces 1O2via the PDT pathway by using endogenous O2. The most remarkable feature of the present nanoformulation is the cooperativity in its therapeutic action, wherein O2 produced during the CDT pathway was used by the 1/ß-CD+ sheet for improving its PDT efficacy in the hypoxic tumor microenvironment. This work represents a unique combination of CDT and PDT for targeted cancer therapy, wherein the CDT action of the nanoagent enhances the PDT efficacy and we strongly believe that this approach would encourage researchers to design similar combination therapy for advancements in the treatment of cancer.

Multifunctional siRNA/ferrocene/cyclodextrin nanoparticles for enhanced chemodynamic cancer therapy.

The therapeutic outcome of chemodynamic therapy (CDT) is greatly hindered by the presence of oxidative damage repair proteins (MTH1) inside cancer cells. These oxidative damage repair proteins detoxify the action of radicals generated by Fenton or Fenton-like reactions. Hence, it is extremely important to develop a simple strategy for the downregulation of MTH1 protein inside cancer cells along with the delivery of metal ions into cancer cells. A one-pot host-guest supramolecular approach for the codelivery of MTH1 siRNA and metal ions into a cancer cell is reported. Our approach involves the fabrication of an inclusion complex between cationic ß-cyclodextrin and a ferrocene prodrug, which spontaneously undergoes amphiphilicity-driven self-assembly to form spherical nanoparticles (NPs) having a positively charged surface. The cationic surface of the NPs was then explored for the loading of MTH1 siRNA through electrostatic interactions. Using HeLa cells as a representative example, efficient uptake of the NPs, delivery of MTH1 siRNA and the enhanced CDT of the nanoformulation are demonstrated. This work highlights the potential of the supramolecular approach as a simple yet efficient method for the delivery of siRNA across the cell membrane for enhanced chemodynamic therapy.

Gross anatomy of vascular supply and drainage of mammary fat pads in mice models.

This work extensively studied the vasculature of mice mammary fat pads (BALB/c and C57BL/6) with special reference to haematogenous drainage routes. Mammary fat pads were five pairs (first cervical, second and third thoracic, fourth abdominal and fifth inguinal), bilaterally symmetrical, extending laterally and continuously with the subcutaneous fascia. The superficial cervical artery and vein primarily accomplished the blood vasculature of the first mammary fat pad, while the lateral thoracic and external thoracic arteries and veins supplied the second and third mammary fat pads. The superficial cervical vein (found parallel to the superficial cervical artery) drained into the external jugular vein. The lateral thoracic artery and external thoracic artery branched almost at the same level as the axillary artery (branch of subclavian artery), the latter being more medial in position. However, in some specimens, the branching of both arteries appeared to be at the same level, and their origins were indistinguishable. The lateral thoracic vein that was parallel to the lateral thoracic artery drained to the axillary vein close to the drainage of the external thoracic vein. The lateral thoracic, superficial caudal epigastric, iliolumbar and external thoracic arteries and veins vascularized the fourth mammary fat pad and displayed anastomosis among themselves. The iliolumbar vein (found parallel to the iliolumbar artery) drained into the inferior vena cava. The superficial caudal epigastric vein (found parallel to the superficial caudal epigastric artery (SCaEA)) drained into the femoral vein. Unlike humans, the internal thoracic artery and vein did not participate in the vasculature of mammary fat pads. The SCaEA and vein supplied blood and drained the fifth mammary fat pad. The anatomical continuity of the fourth and fifth mammary fat pads provided common drainage for both mammary fat pads. The BALB/c and C57BL/6 mice strains studied did not differ in topography and size of mammary fat pads. The vascular supply and drainage of the mammary fat pads also did not differ in the strains studied. Only minor variations could be noted in the small veins draining into the lateral thoracic vein. Lateral tributaries seen in the terminal end of the lateral thoracic vein were absent in the C57BL/6 mice.

19F NMR ON/OFF nanoparticles: a universal approach for the specific detection of DNA-binding cancer biomarkers.

A supramolecular approach for the design of assembly-disassembly-driven 19F ON/OFF nanoparticles, triggered by specific molecular recognition, for the detection of DNA binding cancer biomarkers is reported. The key to our design strategy is the characteristic 19F NMR signal of the probe, which completely vanishes in the aggregated state due to the shortening of T2 relaxation. However, molecular recognition of DNA by the cancer biomarkers through specific molecular recognition results in the disassembly of the nanoparticles, which causes the restoration of the characteristic 19F signal of the probe. The universal nature of the approach is demonstrated through the selective detection of various cancer biomarkers including miRNA, ATP, thrombin, and telomerase.

DNA-directed artificial light-harvesting antenna.

Designing and constructing multichromophoric, artificial light-harvesting antennas with controlled interchromophore distances, orientations, and defined donor-acceptor ratios to facilitate efficient unidirectional energy transfer is extremely challenging. Here, we demonstrate the assembly of a series of structurally well-defined artificial light-harvesting triads based on the principles of structural DNA nanotechnology. DNA nanotechnology offers addressable scaffolds for the organization of various functional molecules with nanometer scale spatial resolution. The triads are organized by a self-assembled seven-helix DNA bundle (7HB) into cyclic arrays of three distinct chromophores, reminiscent of natural photosynthetic systems. The scaffold accommodates a primary donor array (Py), secondary donor array (Cy3) and an acceptor (AF) with defined interchromophore distances. Steady-state fluorescence analyses of the triads revealed an efficient, stepwise funneling of the excitation energy from the primary donor array to the acceptor core through the intermediate donor. The efficiency of excitation energy transfer and the light-harvesting ability (antenna effect) of the triads was greatly affected by the relative ratio of the primary to the intermediate donors, as well as on the interchromophore distance. Time-resolved fluorescence analyses by time-correlated single-photon counting (TCSPC) and streak camera techniques further confirmed the cascading energy transfer processes on the picosecond time scale. Our results clearly show that DNA nanoscaffolds are promising templates for the design of artificial photonic antennas with structural characteristics that are ideal for the efficient harvesting and transport of energy.

Blue-emissive two-component supergelator with aggregation-induced enhanced emission.

Two-component organogels offer several advantages over one-component gels, but their design is highly challenging. Hence, it is extremely important to design new approaches for the crafting of two-component organogels with interesting optical and mechanical properties. Herein, we report the design of a new class of two-component supergelators obtained from the assembly between acid functionalized tetraphenylethylene (TPE)-based dendrons and alkylated melamine. No gelation behaviour is observed for the individual components, but interestingly, remarkable gelation behaviour is observed for their hydrogen-bonded complex. The primary driving force responsible for the gelation is the strong π-π stacking interaction of TPE units. Because of the strong π-stacking of TPEs in the gel state, the C(sp2)-C(sp2) bond rotation of the TPE segment is completely arrested in the gel state, which results in intense fluorescence emission of the gels. Furthermore, excellent elastic response is observed for the gels as evident from their high storage modulus compared to loss modulus values. Our results clearly demonstrate that by the appropriate selection of the molecular components, this approach can be applied for the creation of functional nanomaterials with emergent properties absent in the individual blocks.