Macrocyclization of a Class of Camptothecin Analogues into Tubular Supramolecular Polymers.

Nanostructured supramolecular polymers (SPs) are filamentous assemblies possessing a high degree of internal order and have important uses in regenerative medicine, drug delivery, and soft matter electronics. Despite recent advances in functional SPs, a challenging topic is the development of robust assembly protocols enabling the incorporation of various functional units without altering its supramolecular architecture. We report here the robust tubular assembly of camptothecin (CPT) analogues into functional SPs. Covalent linkage of two CPT moieties to various short hydrophilic segments (e.g., nonionic, cationic, anionic, and zwitterionic) leads to a class of CPT analogues that self-assemble in water into tubular SPs. Systemic administration of nonionic SPs effectively suppresses tumor growth. Furthermore, these tubular SPs act as universal dispersing agents in water for low-molecular-weight hydrophobes.

Comparative levels of carbonyl delivery between mass-market cigars and cigarettes.

The recent 2016 deeming of cigars by the US Food and Drug Administration (FDA) has led to increased interest in cigar science, including ways to accurately measure the harmful and potentially harmful constituents (HPHCs) found within mainstream cigar smoke. At present, there are standardized methods for evaluating HPHCs in mainstream cigarette smoke but none specific to cigar analysis except for nicotine and carbon monoxide. This study sought to analyze carbonyl delivery in marketed cigars and cigarillos and compare them against levels found in cigarettes. To accomplish this the standard cigarette method, CORESTA recommended method 74 (CRM-74), was optimized for cigar smoking including an evaluation of the trapping efficiency and the stability of the carbonyl-hydrazone adducts due to the increased smoke time required for cigar collection. On a per product basis, carbonyl delivery from cigars smoked under CRM-64 conditions was found to yield similar levels of formaldehyde and greater levels of acetaldehyde, acrolein and crotonaldehyde than measured in mainstream cigarette smoke collected under conditions prescribed under ISO standard 3308. Furthermore, on a per product basis, cigarettes smoked under the ISO 20778 intense smoking regime delivered higher levels of formaldehyde, acrolein and crotonaldehyde as compared to cigars smoked under the CORESTA regime, while acetaldehyde was found to be higher in mainstream cigar smoke. Given the recent deeming, this work expands upon previously reported work, limited in scope by either number of products or analytes reported, through the analysis of carbonyl delivery found in the mainstream smoke for 12 brands of cigars and cigarillos.

Self-assembling prodrugs.

Covalent modification of therapeutic compounds is a clinically proven strategy to devise prodrugs with enhanced treatment efficacies. This prodrug strategy relies on the modified drugs that possess advantageous pharmacokinetic properties and administration routes over their parent drug. Self-assembling prodrugs represent an emerging class of therapeutic agents capable of spontaneously associating into well-defined supramolecular nanostructures in aqueous solutions. The self-assembly of prodrugs expands the functional space of conventional prodrug design, affording a possible pathway to more effective therapies as the assembled nanostructure possesses distinct physicochemical properties and interaction potentials that can be tailored to specific administration routes and disease treatment. In this review, we will discuss the various types of self-assembling prodrugs in development, providing an overview of the methods used to control their structure and function and, ultimately, our perspective on their current and future potential.

Molecular design and synthesis of self-assembling camptothecin drug amphiphiles.

The conjugation of small molecular hydrophobic anticancer drugs onto a short peptide with overall hydrophilicity to create self-assembling drug amphiphiles offers a new prodrug strategy, producing well-defined, discrete nanostructures with a high and quantitative drug loading. Here we show the detailed synthesis procedure and how the molecular structure can influence the synthesis of the self-assembling prodrugs and the physicochemical properties of their assemblies. A series of camptothecin-based drug amphiphiles were synthesized via combined solid- and solution-phase synthetic techniques, and the physicochemical properties of their self-assembled nanostructures were probed using a number of imaging and spectroscopic techniques. We found that the number of incorporated drug molecules strongly influences the rate at which the drug amphiphiles are formed, exerting a steric hindrance toward any additional drugs to be conjugated and necessitating extended reaction time. The choice of peptide sequence was found to affect the solubility of the conjugates and, by extension, the critical aggregation concentration and contour length of the filamentous nanostructures formed. In the design of self-assembling drug amphiphiles, the number of conjugated drug molecules and the choice of peptide sequence have significant effects on the nanostructures formed. These observations may allow the fine-tuning of the physicochemical properties for specific drug delivery applications, ie systemic vs local delivery.

Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

Self-healable, tough and highly stretchable ionic nanocomposite physical hydrogels.

We present a facile strategy to synthesize self-healable tough and highly stretchable hydrogels. Our design rationale for the creation of ionic cross-linked hydrogels is to graft an acrylic acid monomer on the surface of vinyl hybrid silica nanoparticles (VSNPs) for the growth of poly(acrylic) acid (PAA), and the obtained VSNP-PAA nanobrush can be used as a gelator. Physical cross-linking through hydrogen bonding and ferric ion-mediated ionic interactions between PAA polymer chains of the gelators yielded ionic nanocomposite physical hydrogels with excellent and balanced mechanical properties (tensile strength 860 kPa, elongation at break ∼2300%), and the ability to self-repair (tensile strength ∼560 kPa, elongation at break ∼1800%). The toughness and stretchability arise from the reversible cross-linking interactions between the polymer chains that help dissipate energy through stress (deformation) triggered dynamic processes. These unique properties will enable greater application of these hydrogel materials, especially in tissue engineering.

Amino acid sequence in constitutionally isomeric tetrapeptide amphiphiles dictates architecture of one-dimensional nanostructures.

The switching of two adjacent amino acids can lead to differences in how proteins fold thus affecting their function. This effect has not been extensively explored in synthetic peptides in the context of supramolecular self-assembly. Toward this end, we report here the use of isomeric peptide amphiphiles as molecular building blocks to create one-dimensional (1D) nanostructures. We show that four peptide amphiphile isomers, with identical composition but a different sequence of their four amino acids, can form drastically different types of 1D nanostructures under the same conditions. We found that molecules with a peptide sequence of alternating hydrophobic and hydrophilic amino acids such as VEVE and EVEV self-assemble into flat nanostructures that can be either helical or twisted. On the other hand, nonalternating isomers such as VVEE and EEVV result in the formation of cylindrical nanofibers. Furthermore, we also found that when the glutamic acid is adjacent to the alkyl tail the supramolecular assemblies appear to be internally flexible compared to those with valine as the first amino acid. These results clearly demonstrate the significance of peptide side chain interactions in determining the architectures of supramolecular assemblies.

Enhanced cellular entry and efficacy of tat conjugates by rational design of the auxiliary segment.

Conjugation with a cell penetrating peptide such as Tat presents an effective approach to improve the intracellular accumulation of molecules with low membrane permeability. This strategy, however, leads to a reduced cellular entry of molecules that can cross cell membrane effectively. We report here that covalent linkage of an additional hydrophobic unit that mimics a hydrophobic domain near the Tat sequence can further improve the cellular uptake of the parental conjugate into cancer cells regardless of the membrane permeability of the unconjugated molecule. Both fluorescent imaging and flow cytometry measurements confirmed the effect of palmitoylation on the increased internalization of the Tat conjugates with either 5-carboxyfluorescein (5-FAM), a nonmembrane penetrating dye, or doxorubicin, an anticancer cancer drug that can readily diffuse across cell membranes. In the case of the Tat-doxorubicin conjugate, palmitoylation improves the conjugate's anticancer activity in both drug sensitive and resistant cervical cancer cell lines. We further demonstrate that modification of a Tat-5-FAM conjugate with a hydrophobic quencher could not only efficiently quench the fluorescence outside of cancer cell but also facilitate its entry into MCF-7 breast cancer cells. These results highlight the importance of rational molecular design of using peptide conjugation chemistry in cancer therapeutics and diagnostics.

Rational design of MMP degradable peptide-based supramolecular filaments.

One-dimensional nanostructures formed by self-assembly of small molecule peptides have been extensively explored for use as biomaterials in various biomedical contexts. However, unlike individual peptides that can be designed to be specifically degradable by enzymes/proteases of interest, their self-assembled nanostructures, particularly those rich in ß-sheets, are generally resistant to enzymatic degradation because the specific cleavage sites are often embedded inside the nanostructures. We report here on the rational design of ß-sheet rich supramolecular filaments that can specifically dissociate into less stable micellar assemblies and monomers upon treatment with matrix metalloproteases-2 (MMP-2). Through linkage of an oligoproline segment to an amyloid-derived peptide sequence, we first synthesized an amphiphilic peptide that can undergo a rapid morphological transition in response to pH variations. We then used MMP-2 specific peptide substrates as multivalent cross-linkers to covalently fix the amyloid-like filaments in the self-assembled state at pH 4.5. Our results show that the cross-linked filaments are stable at pH 7.5 but gradually break down into much shorter filaments upon cleavage of the peptidic cross-linkers by MMP-2. We believe that the reported work presents a new design platform for the creation of amyloid-like supramolecular filaments responsive to enzymatic degradation.

Supramolecular nanostructures formed by anticancer drug assembly.

We report here a supramolecular strategy to directly assemble the small molecular hydrophobic anticancer drug camptothecin (CPT) into discrete, stable, well-defined nanostructures with a high and quantitative drug loading. Depending on the number of CPTs in the molecular design, the resulting nanostructures can be either nanofibers or nanotubes, and have a fixed CPT loading content ranging from 23% to 38%. We found that formation of nanostructures provides protection for both the CPT drug and the biodegradable linker from the external environment and thus offers a mechanism for controlled release of CPT. Under tumor-relevant conditions, these drug nanostructures can release the bioactive form of CPT and show in vitro efficacy against a number of cancer cell lines. This strategy can be extended to construct nanostructures of other types of anticancer drugs and thus presents new opportunities for the development of self-delivering drugs for cancer therapeutics.

Cellular uptake and cytotoxicity of drug-peptide conjugates regulated by conjugation site.

Conjugation of anticancer drugs to hydrophilic peptides such as Tat is a widely adopted strategy to improve the drug's solubility, cellular uptake, and potency against cancerous cells. Here we report that attachment of an anticancer drug doxorubicin to the N- or C-terminal of the Tat peptide can have a significant impact on their cellular uptake and cytotoxicity against both drug-sensitive and drug-resistant cancer cells. We observed higher cellular uptake by both cell lines for C-terminal conjugate relative to the N-terminal analogue. Our results reveal that the C-terminal conjugate partially overcame the multidrug resistance of cervical cancer cells, while the N-terminal conjugate showed no significant improvement in cytotoxicity when compared with free doxorubicin. We also found that both N- and C-conjugates offer a mechanism to circumvent drug efflux associated with multidrug resistance.

One-step fabrication of self-assembled peptide thin films with highly dispersed noble metal nanoparticles.

Fabrication of organic thin films with highly dispersed inorganic nanoparticles is a very challenging topic. In this work, a new approach that combines electron-induced molecular self-assembly with simultaneous nanoparticle formation by room temperature electron reduction was developed to prepare peptide thin films with highly dispersed noble metal nanoparticles. Argon glow discharge was employed as the resource of electrons. The peptide motif KLVFF (Aß16-20) self-assembled into two-dimensional membranes under the influence of hydrated electrons, while the metal ions in solution can be simultaneously reduced by electrons to form nanoparticles. Our TEM imaging reveals that metal nanoparticles were well-distributed in the resulting peptide thin films. Our results also suggest that the size of metal nanoparticles can be tuned by varying the initial concentration of the metal ion. This simple approach can be viewed as a promising strategy to create hybrid thin films that integrate functional inorganics into biomolecule scaffolds.

Analysis and differentiation of tobacco-derived and synthetic nicotine products: Addressing an urgent regulatory issue.

There is significant regulatory and economic need to distinguish analytically between tobacco-derived nicotine (TDN) and synthetic nicotine (SyN) in commercial products. Currently, commercial e-liquid and oral pouch products are available that contain tobacco-free nicotine, which could be either extracted from tobacco or synthesized. While tobacco products that contain TDN are regulated by FDA Center for Tobacco Products, those with SyN are currently not in the domain of any regulatory authority. This regulatory difference provides an economic incentive to use or claim the use of SyN to remain on the market without submitting a Premarket Tobacco Product Application. TDN is ~99.3% (S)-nicotine, whereas SyN can vary from racemic (50/50 (R)/(S)) to ≥ 99% (S)-nicotine, i.e., chemically identical to the tobacco-derived compound. Here we report efforts to distinguish between TDN and SyN in various samples by characterizing impurities, (R)/(S)-nicotine enantiomer ratio, (R)/(S)-nornicotine enantiomer ratio, and carbon-14 (14C) content. Only 14C analysis accurately and precisely differentiated TDN (100% 14C) from SyN (35-38% 14C) in all samples tested. 14C quantitation of nicotine samples by accelerator mass spectrometry is a reliable determinate of nicotine source and can be used to identify misbranded product labelled as containing SyN. This is the first report to distinguish natural, bio-based nicotine from synthetic, petroleum-based nicotine across a range of pure nicotine samples and commercial e-liquid products.

Nature-inspired delivery of mitochondria-targeted angiotensin receptor blocker.

Mitochondria are critical regulators of cellular function and survival. We have previously demonstrated that functional angiotensin receptors embedded within the inner mitochondrial membrane modulate mitochondrial energy production and free radical generation. The expression of mitochondrial angiotensin II type-1 receptors increases during aging, with a complementary decrease in angiotensin II type-2 receptor density. To address this age-associated mitochondrial dysfunction, we have developed a mitochondria-targeted delivery system to effectively transport angiotensin type-1 receptor blocker-Losartan (mtLOS) into the inner mitochondrial membrane. We engineered mtLOS to become active within the mitochondria after cleavage by mitochondrial peptidases. Our data demonstrate effective and targeted delivery of mtLOS into the mitochondria, compared to a free Losartan, or Losartan conjugated to a scrambled mitochondrial target signal peptide, with significant shifts in mitochondrial membrane potential upon mtLOS treatment. Furthermore, engineered mitochondrial-targeting modalities could open new avenues to transport nonmitochondrial proteins into the mitochondria, such as other macromolecules and therapeutic agents.

Synergistic antitumor activity of a self-assembling camptothecin and capecitabine hybrid prodrug for improved efficacy.

The direct use of anticancer drugs to create their own nanostructures is an emerging concept in the field of drug delivery to obtain nanomedicines of high drug loading and high reproducibility, and the combination use of two or more drugs has been a proven clinical strategy to enhance therapeutic outcomes. We report here the synthesis, assembly and cytotoxicity evaluation of self-assembling hybrid prodrugs containing both camptothecin (CPT) and a capecitabine (Cap) analogue. CPT and Cap molecules were conjugated onto a short ß-sheet-forming peptide (Sup35) to yield three different self-assembling prodrugs (dCPT-Sup35, CPT-Cap-Sup35 and dCap-Sup35). We found that the chemical structure of conjugated drugs could strongly influence their assembled morphology as well as their structural stability in aqueous solution. With a decrease in number of CPT units, the resulting nanostructures exhibited a morphological transformation from nanofibers (dCPT-Sup35) to filaments (CPT-Cap-Sup35) then to spherical particles (dCap-Sup35). Notably, the hybrid CPT-Cap prodrug showed a synergistic effect and significantly enhanced potency against three esophageal adenocarcinoma cell lines compared with the two homo-prodrugs (dCPT-Sup35 and dCap-Sup35) as well as free parent drugs (CPT, 5-Fu and CPT/5-FU mixture (1:1)). We believe this work represents a conceptual advancement in integrating two structurally distinct drugs of different action mechanisms into a single self-assembling hybrid prodrug to construct self-deliverable nanomedicines for more effective combination chemotherapy.

Peptide-drug conjugates as effective prodrug strategies for targeted delivery.

Peptide-drug conjugates (PDCs) represent an important class of therapeutic agents that combine one or more drug molecules with a short peptide through a biodegradable linker. This prodrug strategy uniquely and specifically exploits the biological activities and self-assembling potential of small-molecule peptides to improve the treatment efficacy of medicinal compounds. We review here the recent progress in the design and synthesis of peptide-drug conjugates in the context of targeted drug delivery and cancer chemotherapy. We analyze carefully the key design features in choosing the peptide sequence and linker chemistry for the drug of interest, as well as the strategies to optimize the conjugate design. We highlight the recent progress in the design and synthesis of self-assembling peptide-drug amphiphiles to construct supramolecular nanomedicine and nanofiber hydrogels for both systemic and topical delivery of active pharmaceutical ingredients.

Isomeric control of the mechanical properties of supramolecular filament hydrogels.

Supramolecular filament hydrogels are an emerging class of biomaterials that hold great promise for regenerative medicine, tissue engineering, and drug delivery. However, fine-tuning of their bulk mechanical properties at the molecular level without altering their network structures remains a significant challenge. Here we report an isomeric strategy to construct amphiphilic peptides through the conjugation of isomeric hydrocarbons to influence the local viscoelastic properties of their resulting supramolecular hydrogels. In this case, the packing requirements of the chosen isomeric hydrocarbons within the supramolecular filaments are dictated by their atomic arrangements at the molecular and intermolecular levels. Atomistic molecular dynamics simulations suggest that this design strategy can subtly alter the molecular packing at the interface between the peptide domain and the hydrophobic core of the supramolecular assemblies, without changing both the filament width and morphology. Our results from wide-angle X-ray scattering and molecular simulations further confirm that alterations to the intermolecular packing at the interface impact the strength and degree of hydrogen bonding within the peptide domains. This subtle difference in the isomeric hydrocarbon design and their consequent packing difference led to variations in the persistence length of the individual supramolecular filaments. Microrheological analysis reveals that this difference in filament stiffness enables the fine-tuning of the mechanical properties of the hydrogel at the macroscopic scale. We believe that this isomeric platform provides an innovative method to tune the local viscoelastic properties of supramolecular polymeric hydrogels without necessarily altering their network structures.

Drug-Bearing Supramolecular Filament Hydrogels as Anti-Inflammatory Agents.

We report here on the covalent conversion of the anti-inflammatory agent ketoprofen into self-assembling prodrugs that enable the effective purification of ketoprofen enantiomers, the improved selectivity and potency of ketoprofen, as well as the formation of one-component drug-bearing supramolecular hydrogels. We found that the ketoprofen hydrogelator could exhibit much-enhanced selectivity for cyclooxygenase 2 (COX-2) over COX-1, reduce the concentration of inflammatory cytokines (IL-1 and TNFα), and induce apoptosis in fibroblast-like synoviocytes while maintaining biocompatibility with healthy chondrocytes. In addition, these anti-inflammatory agent-containing hydrogels demonstrated the ability to retain the therapeutic within a joint cavity after intra-articular injection, exhibiting a slow, steady release into the plasma. We believe that upon further optimization these drug-based injectable supramolecular hydrogels could provide the basis for a local treatment strategy for rheumatoid arthritis and similar conditions.

Is stress-only imaging practical when a 1-day stress-rest 99mTc-tetrofosmin protocol is used?

OBJECTIVES: To investigate whether a significant number of normal 1-day stress-rest 99mTc-tetrofosmin myocardial perfusion scintigraphy (MPS) studies can be identified from the low-dose stress acquisition alone, and whether technical staff can find such studies reliably. METHODS: The supervising consultant and four technologists independently graded the stress acquisitions from 200 consecutive MPS studies using a five-point scale. Studies were classified as normal or abnormal according to the final clinical report based on the completed stress-rest protocol. RESULTS: Between 31 and 62 studies (16-31%) were classified as definitely normal from the stress acquisition alone, of which 0-4 (0-9%) proved abnormal on the final report. Of stress studies graded definitely normal by each technologist, the consultant disagreed in 13-34% of cases. Of 78 stress studies graded definitely normal by at least one technologist, 6% turned out to be abnormal and the consultant disagreed in 33%. When there was agreement between at least two technologists (57 studies), the rates fell to 4% and 21% respectively. CONCLUSIONS: Technologists could make the decision to avoid a resting study in up to 30% of patients following a normal low-dose stress acquisition. The supervising nuclear cardiologist would disagree in perhaps one in five cases, even when there was consensus between two or more technologists. These patients would suffer minor inconvenience by being recalled for a rest acquisition on a second day, but there would be important savings in time and radiation exposure for the majority.

Building Nanostructures with Drugs.

The convergence of nanoscience and drug delivery has prompted the formation of the field of nanomedicine, one that exploits the novel physicochemical and biological properties of nanostructures for improved medical treatments and reduced side effects. Until recently, this nanostructure-mediated strategy considered the drug to be solely a biologically active compound to be delivered, and its potential as a molecular building unit remained largely unexplored. A growing trend within nanomedicine has been the use of drug molecules to build well-defined nanostructures of various sizes and shapes. This strategy allows for the creation of self-delivering supramolecular nanomedicines containing a high and fixed drug content. Through rational design of the number and type of the drug incorporated, the resulting nanostructures can be tailored to assume various morphologies (e.g. nanospheres, rods, nanofibers, or nanotubes) for a particular mode of administration such as systemic, topical, and local delivery. This review covers the recent advances in this rapidly developing field, with the aim of providing an in-depth evaluation of the exciting opportunities that this new field could create to improve the current clinical practice of nanomedicine.