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.

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.

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.

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.

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.

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.

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.

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.

Supramolecular Crafting of Self-Assembling Camptothecin Prodrugs with Enhanced Efficacy against Primary Cancer Cells.

Chemical modification of small molecule hydrophobic drugs is a clinically proven strategy to devise prodrugs with enhanced treatment efficacy. While this prodrug strategy improves the parent drug's water solubility and pharmacokinetic profile, it typically compromises the drug's potency against cancer cells due to the retarded drug release rate and reduced cellular uptake efficiency. Here we report on the supramolecular design of self-assembling prodrugs (SAPD) with much improved water solubility while maintaining high potency against cancer cells. We found that camptothecin (CPT) prodrugs created by conjugating two CPT molecules onto a hydrophilic segment can associate into filamentous nanostructures in water. Our results suggest that these SAPD exhibit much greater efficacy against primary brain cancer cells relative to that of irinotecan, a clinically used CPT prodrug. We believe these findings open a new avenue for rational design of supramolecular prodrugs for cancer treatment.

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.

Targeting Tumors with Small Molecule Peptides.

Chemotherapeutic treatment of cancers is a challenging endeavor, hindered by poor selectivity towards tumorous tissues over healthy ones. Preferentially delivering a given drug to tumor sites necessitates the use of targeting elements, of which there are a wide range in development. In this Review, we highlight recent examples of peptide-based targeting ligands that have been exploited to selectively deliver a chemotherapeutic payload to specific tumor-associated sites such as the vasculature, lymphatics, or cell surface. The advantages and limitations of such approaches will be discussed with a view to potential future development. Additionally, we will also examine how peptide-based ligands can be used diagnostically in the detection and characterization of cancers through their incorporation into imaging agents.

Electrostatic-Driven Lamination and Untwisting of ß-Sheet Assemblies.

Peptides or peptide conjugates capable of assembling into one-dimensional (1D) nanostructures have been extensively investigated over the past two decades due to their implications in human diseases and also their interesting applications as biomaterials. While many of these filamentous assemblies contain a ß-sheet-forming sequence as the key design element, their eventual morphology could assume a variety of shapes, such as fibrils, ribbons, belts, or cylinders. Deciphering the key factors that govern the stacking fashion of individual ß-sheets will help understand the polymorphism of peptide assemblies and greatly benefit the development of functional materials from customized molecular design. Herein, we report the decisive role of electrostatic interactions in the lamination and untwisting of 1D assemblies of short peptides. We designed and synthesized three short peptides containing only six amino acids (EFFFFE, KFFFFK, and EFFFFK) to elucidate the effective control of ß-sheet stacking. Our results clearly suggest that electrostatic repulsions between terminal charges reduce the pitch of the twisting ß-sheet tapes, thus leading to highly twisted, intertwined fibrils or twisted ribbons, whereas reducing this repulsion, either through molecular design of peptide with opposite terminal charges or through coassembly of two peptides carrying opposite charges, results in formation of infinite assemblies such as belt-like morphologies. We believe these observations provide important insight into the generic design of ß-sheet assemblies.

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.

Activatable nanoprobes for biomolecular detection.

Precise detection of pathologically relevant biomolecules could provide essential information on important intercellular, cellular, and subcellular events for accurate disease diagnosis and staging, thus leading to appropriate treatment recommendation. Activatable nanoprobes are nanoscale objects that can be turned on through specific reactions or interactions with biomolecules of interest, and afford some advantageous properties for improved detection of biomolecules both in vitro and in vivo. In this brief review, we highlight several recent examples in the development of activatable nanoprobes for biomolecule detection.

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.

Multiwalled nanotubes formed by catanionic mixtures of drug amphiphiles.

Mixing of oppositely charged amphiphilic molecules (catanionic mixing) offers an attractive strategy to produce morphologies different from those formed by individual molecules. We report here on the use of catanionic mixing of anticancer drug amphiphiles to construct multiwalled nanotubes containing a fixed and high drug loading. We found that the molecular mixing ratio, the solvent composition, the overall drug concentrations, as well as the molecular design of the studied amphiphiles are all important experimental parameters contributing to the tubular morphology. We believe these results demonstrate the remarkable potential that anticancer drugs could offer to self-assemble into discrete nanostructures and also provide important insight into the formation mechanism of nanotubes by catanionic mixtures. Our preliminary animal studies reveal that the CPT nanotubes show significantly prolonged retention time in the tumor site after intratumoral injection.

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.

Controlled release of free doxorubicin from peptide-drug conjugates by drug loading.

Covalent modification of a drug with a peptide moiety has been extensively used as an effective strategy to improve the drug's therapeutic outcome. One important consideration in the design of such a prodrug is the release of the free drug from the covalently bound form in a desired fashion. In most cases, the free drug release rate is controlled by the use of various chemical linkers that bridge the drug to the auxiliary segment. We report here that the degree of drug conjugation per peptide could also regulate the drug release in addition to its apparent effect on drug loading of the resulting conjugates. In this work, we synthesized three peptide-drug conjugates (NTD, d-NTD and q-NTD) in which the cell penetrating peptide Tat is covalently connected to one, two, or four doxorubicin, respectively, through a cathepsin B degradable tetrapeptide linker (-Gly-Phe-Leu-Gly-). We found that the number of doxorubicin within the conjugate impacts the release of doxorubicin in a significant way, with q-NTD showing the slowest release rate while NTD showing the fastest release rate. Our cellular uptake experiments reveal that q-NTD accumulated most effectively within cancer cells while NTD shows the lowest intracellular accumulation concentration. Interestingly, our cell viability assessment using a SRB assay reveals that d-NTD is the most potent conjugate against HepG2 human liver cancer cells. These results suggest that intracellular accumulation efficiency and the free drug release rate are two important factors that determine the in vitro efficacy of drug conjugates. To further validate this conclusion, we conjugated a short hydrocarbon onto the NTD to improve its cellular uptake, and found that the resulting conjugate, C16NTD, exhibited comparable intracellular accumulation as the q-NTD conjugate but superior anticancer activity due to its more effective release of free doxorubicin.