Development of bioconjugate-based delivery systems for nucleic acids.

Nucleic acids are a class of drugs that can modulate gene and protein expression by various mechanisms, namely, RNAi, mRNA degradation by RNase H cleavage, splice modulation, and steric blocking of protein binding or mRNA translation, thus exhibiting immense potential to treat various genetic and rare diseases. Unlike protein-targeted therapeutics, the clinical use of nucleic acids relies on Watson-Crick sequence recognition to regulate aberrant gene expression and impede protein translation. Though promising, targeted delivery remains a bottleneck for the clinical adoption of nucleic acid-based therapeutics. To overcome the delivery challenges associated with nucleic acids, various chemical modifications and bioconjugation-based delivery strategies have been explored. Currently, liver targeting by N-acetyl galactosamine (GalNAc) conjugation has been at the forefront for the treatment of rare and various metabolic diseases, which has led to FDA approval of four nucleic acid drugs. In addition, various other bioconjugation strategies have been explored to facilitate active organ and cell-enriched targeting. This review briefly covers the different classes of nucleic acids, their mechanisms of action, and their challenges. We also elaborate on recent advances in bioconjugation strategies in developing a diverse set of ligands for targeted delivery of nucleic acid drugs.

Adsorption and Distribution of Triplex-Hybridizing Bioconjugated Gold Nanoclusters Inside Spermatozoa- A Study Using Confocal Microscopy and Fluorescence Lifetime Imaging.

The study of the interactions between biofunctionalized gold nanoclusters (Au NCs) and spermatozoa is highly relevant to evaluate the potential of Au NCs as imaging probes and transfection agents in reproductive biology. In this work, confocal laser scanning microscopy (CLSM) was used to investigate the distribution of Au NCs bioconjugated with peptide (nuclear localisation sequence, NLS) and oligonucleotide (locked nucleic acid, LNA) ligands in bovine spermatozoa. Fluorescence lifetime imaging (FLIM) was employed to detect changes in the NC's chemical environment. We observed a pronounced regio-selective accumulation of the bioconjugates in spermatozoa with high concentration at the equatorial segment. Furthermore, 3D-CLSM showed successful non-endosomal cellular uptake of the conjugates by intact sperm cells and the distribution of the bioconjugates was found to be influenced by the ligand types. Interestingly, the FLIM data showed differences in lifetime depending on membrane integrity. Furthermore, ligand-dependent changes in lifetime between NC bioconjugates carrying peptide and oligonucleotide ligands were found, probably attributed to specific interactions with sperm cell compartments.

Luminescent photon-upconversion nanoparticles with advanced functionalization for smart sensing and imaging.

Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics.

Analgesic Peptides: From Natural Diversity to Rational Design.

Pain affects one-third of the global population and is a significant public health issue. The use of opioid drugs, which are the strongest painkillers, is associated with several side effects, such as tolerance, addiction, overdose, and even death. An increasing demand for novel, safer analgesic agents is a driving force for exploring natural sources of bioactive peptides with antinociceptive activity. Since the G protein-coupled receptors (GPCRs) play a crucial role in pain modulation, the discovery of new peptide ligands for GPCRs is a significant challenge for novel drug development. The aim of this review is to present peptides of human and animal origin with antinociceptive potential and to show the possibilities of their modification, as well as the design of novel structures. The study presents the current knowledge on structure-activity relationship in the design of peptide-based biomimetic compounds, the modification strategies directed at increasing the antinociceptive activity, and improvement of metabolic stability and pharmacodynamic profile. The procedures employed in prolonged drug delivery of emerging compounds are also discussed. The work summarizes the conditions leading to the development of potential morphine replacements.

Lactoferricin B Combined with Antibiotics Exhibits Leukemic Selectivity and Antimicrobial Activity.

The fusion of penetrating peptides (PPs), e.g., cell penetration peptides (CPPs) or antimicrobial peptides (AMPs), together with antimicrobial agents is an expanding research field. Specific AMPs, such as lactoferricin B (LfcinB), have demonstrated strong antibacterial, antifungal, and antiparasitic activity, as well as valuable anticancer activity, proving beneficial in the development of anticancer conjugates. The resulting conjugates offer potential dual functionality, acting as both an anticancer and an antimicrobial agent. This is especially necessary in cancer treatment, where microbial infections pose a critical risk. Leukemic cells frequently exhibit altered outer lipid membranes compared to healthy cells, making them more sensitive to compounds that interfere with their membrane. In this study, we revisited and reanalyzed our earlier research on LfcinB and its conjugates. Furthermore, we carried out new experiments with a specific focus on cell proliferation, changes in membrane asymmetric phosphatidylserine location, intracellular reactive oxygen species (ROS) generation, mitochondrial functions, and in vitro bacterial topoisomerase inhibition.

Synthesis and glutathione peroxidase (GPx)-like activity of selenocystine (SeC) bioconjugates of biotin and lipoic acid.

The conjugation of active biomolecules provides insight into their bioreactivity, leading to many applications in biotechnology and materials science. Herein, we report L-selenocystine (SeC) bioconjugates of lipoic acid (universal antioxidant) and biotin (Vitamin-H). The SeC-bioconjugates, SeC-Biotin (1) and SeC-Lipoic acid (2) were synthesized using solid phase peptide synthesis (SPPS) method and were characterized by multinuclear 1D (1H, 13C, 77Se) and 2D (1H-1H COSY and 1H-13C TOCSY) NMR spectroscopy, ESI-MS spectrometry, and RP-HPLC. The GPx-like enzyme mimicking activity of the SeC-bioconjugates 1 and 2 has been investigated through the coupled reductase assay method for the catalytic reductions of hydrogen peroxide into water. A significant enhancement in GPx-like enzymatic activity was observed for both novel bioconjugates SeC-Biotin (1) and SeC-Lipoic acid (2) as compared to diphenyl diselenide (Ph2Se2), L-selenocystine (SeC), biotin, lipoic acid, and ebselen.

A review of biomolecules conjugated lanthanide up-conversion nanoparticles-based fluorescence probes in food safety and quality monitoring applications.

Upconversion nanoparticles (UCNPs) are known to possess unique characteristics, which allow them to overcome a number of issues that plague traditional fluorescence probes. UCNPs have been employed in a variety of applications, but it is arguably in the realm of optical sensors where they have shown the most promise. Biomolecule conjugated UCNPs-based fluorescence probes have been developed to detect and quantify a wide range of analytes, from metal ions to biomolecules, with great specificity and sensitivity. In this review, we have given much emphasis on the recent trends and progress in the preparation strategies of bioconjugated UCNPs and their potential application as fluorescence sensors in the trace level detection of food industry-based toxicants and adulterants. The paper discusses the preparation and functionalisation strategies of commonly used biomolecules over the surface of UCNPs. The use of different sensing strategies namely heterogenous and homogenous assays, underlying fluorescence mechanisms in the detection process of food adulterants are summarized in detail. This review might set a precedent for future multidisciplinary research including the development of novel biomolecules conjugated UCNPs for potential applications in food science and technology.

Oxidation-labile linkers for controlled drug delivery.

The continuous symbiosis throughout chemical biology and drug discovery has led to the design of innovative bifunctional molecules for targeted and controlled drug delivery. Among the different tools, protein-drug and peptide-drug conjugates are trend approaches to achieve targeted delivery, selectivity and efficacy. To meet the main goals of these bioconjugates, the selection of the appropriate payloads and linkers is crucial, as they must provide in vivo stability, while they may also help to achieve the therapeutic target and action. In neurodegenerative diseases or some cancer types, where oxidative stress plays an important role, linkers sensitive to oxidative conditions may be able to release the drug once the conjugate achieves the target. Considering specially this specific application, this mini-review covers the most relevant publications on oxidation-labile linkers.

Peptide Drug Conjugates and Their Role in Cancer Therapy.

Drug conjugates have become a significant focus of research in the field of targeted medicine for cancer treatments. Peptide-drug conjugates (PDCs), a subset of drug conjugates, are composed of carrier peptides ranging from 5 to 30 amino acid residues, toxic payloads, and linkers that connect the payload to the peptide. PDCs are further broken down into cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs), each having their own differences in the delivery of cytotoxic payloads. Generally, PDCs as compared to other drug conjugates-like antibody-drug conjugates (ADCs)-have advantages in tumor penetration, ease of synthesis and cost, and reduced off-target effects. Further, as compared to traditional cancer treatments (e.g., chemotherapy and radiation), PDCs have higher specificity for the target cancer with generally less toxic side effects in smaller doses. However, PDCs can have disadvantages such as poor stability and rapid renal clearance due to their smaller size and limited oral bioavailability due to digestion of its peptide structure. Some of these challenges can be overcome with modifications, and despite drawbacks, the intrinsic small size of PDCs with high target specificity still makes them an attractive area of research for cancer treatments.

A Theoretical Analysis of Interaction Energies and Intermolecular Interactions between Amphotericin B and Potential Bioconjugates Used in the Modification of Nanocarriers for Drug Delivery.

Amphotericin B (AmB) is an antibiotic with a wide spectrum of action and low multidrug resistance, although it exhibits self-aggregation, low specificity, and solubility in aqueous media. An alternative for its oral administration is its encapsulation in polymers modified with bioconjugates. The aim of the present computational research is to determine the affinity between AmB and six bioconjugates to define which one could be more suitable. The CAM-B3LYP-D3/6-31+G(d,p) method was used for all computational calculations. The dimerization enthalpy of the most stable and abundant systems at pH = 7 allows obtaining this affinity order: AmB_1,2-distearoyl-sn-glycerol-3-phosphorylethanolamine (DSPE) > AmB_γ-cyclodextrin > AmB_DSPEc > AmB_retinol > AmB_cholesterol > AmB_dodecanol, where DSPEc is a DSPE analog. Quantum theory of atoms in molecules, the non-covalent interactions index, and natural bond orbital analysis revealed the highest abundance of noncovalent interactions for AmB-DSPE (51), about twice the number of interactions of the other dimers. Depending on the interactions' strength and abundance of the AmB-DSPE dimer, these are classified as strong: O-H---O (2), N-H---O (3) and weak: C-H---O (25), H---H (18), C-H---C (3). Although the C-H---O hydrogen bond is weak, the number of interactions involved in all dimers cannot be underestimated. Thus, non-covalent interactions drive the stabilization of copolymers, and from our analysis, the most promising candidates for encapsulating are DSPE and γ-cyclodextrin.

Antiviral Evaluation of New Synthetic Bioconjugates Based on GA-Hecate: A New Class of Antivirals Targeting Different Steps of Zika Virus Replication.

Re-emerging arboviruses represent a serious health problem due to their rapid vector-mediated spread, mainly in urban tropical areas. The 2013-2015 Zika virus (ZIKV) outbreak in South and Central America has been associated with cases of microcephaly in newborns and Guillain-Barret syndrome. We previously showed that the conjugate gallic acid-Hecate (GA-FALALKALKKALKKLKKALKKAL-CONH2)-is an efficient inhibitor of the hepatitis C virus. Here, we show that the Hecate peptide is degraded in human blood serum into three major metabolites. These metabolites conjugated with gallic acid were synthesized and their effect on ZIKV replication in cultured cells was evaluated. The GA-metabolite 5 (GA-FALALKALKKALKKL-COOH) was the most efficient in inhibiting two ZIKV strains of African and Asian lineage at the stage of both virus entry (virucidal and protective) and replication (post-entry). We also demonstrate that GA-metabolite 5 does not affect cell growth after 7 days of continuous treatment. Thus, this study identifies a new synthetic antiviral compound targeting different steps of ZIKV replication in vitro and with the potential for broad reactivity against other flaviviruses. Our work highlights a promising strategy for the development of new antivirals based on peptide metabolism and bioconjugation.

Synthesis of Indole-Coupled KYNA Derivatives via C-N Bond Cleavage of Mannich Bases.

KYNAs, a compound with endogenous neuroprotective functions and an indole that is a building block of many biologically active compounds, such as a variety of neurotransmitters, are reacted in a transformation building upon Mannich bases. The reaction yields triarylmethane derivatives containing two biologically potent skeletons, and it may contribute to the synthesis of new, specialised neuroprotective compounds. The synthesis has been investigated via two procedures and the results were compared to those of previous studies. A possible alternative reaction route through acid catalysis has been established.

Near-Infrared-Emissive AIE Bioconjugates: Recent Advances and Perspectives.

Near-infrared (NIR) fluorescence materials have exhibited formidable power in the field of biomedicine, benefiting from their merits of low autofluorescence background, reduced photon scattering, and deeper penetration depth. Fluorophores possessing planar conformation may confront the shortcomings of aggregation-caused quenching effects at the aggregate level. Fortunately, the concept of aggregation-induced emission (AIE) thoroughly reverses this dilemma. AIE bioconjugates referring to the combination of luminogens showing an AIE nature with biomolecules possessing specific functionalities are generated via the covalent conjugation between AIEgens and functional biological species, covering carbohydrates, peptides, proteins, DNA, and so on. This perfect integration breeds unique superiorities containing high brightness, good water solubility, versatile functionalities, and prominent biosafety. In this review, we summarize the recent progresses of NIR-emissive AIE bioconjugates focusing on their design principles and biomedical applications. Furthermore, a brief prospect of the challenges and opportunities of AIE bioconjugates for a wide range of biomedical applications is presented.

Optimization of Nanohybrid Biosensors Based on Electro-Crosslinked Tannic Acid Capped Nanoparticles/Enzyme.

Enzymes/Nanoparticles (NPs) bioconjugates are massively used nowadays to develop thin films for optical and electrochemical biosensors. Nevertheless, their full characterization as a thin coating onto electrodes remains little discussed, in particular the influence of NPs size and enzyme/NPs ratio used in the electrodeposition solution. In this study, GOx (160 kDa) and HRP (44 kDa) were used in association with tannic acid capped gold NPs (a series with sizes from 7 to 40 nm) to electrodeposit biosensor coatings, sensitive towards glucose and H2O2, respectively. The electrodeposition process was based on a mussel-inspired electro-crosslinking between gallol moieties of tannic acid (at the surface of NPs) and amine moieties of the enzymes. On one hand, the sensitivity of the GOx/NPs coatings depends strongly on the NP size and the enzyme/NPs molar ratio of the electrodeposition solution. An optimal sensitivity was obtained by electrodeposition of 11 nm NPs at a GOx/NPs molar ratio close to the theoretical value of the enzyme monolayer. On the other hand, a modest influence of the NPs size was found on the sensitivity in the case of the electrodeposited HRP/NPs coatings, reaching a plateau at the HRP/NPs molar ratio close to the value of the theoretical enzyme monolayer. In both cases, the enzyme/NPs molar ratio played a role in the sensitivity. To fully understand the parameters driving the biosensor sensitivity, a comprehensive evaluation of the colloidal state of the bioconjugates is proposed here.

Semisynthetic Derivatives of Pentacyclic Triterpenes Bearing Heterocyclic Moieties with Therapeutic Potential.

Medicinal plants have been used by humans since ancient times for the treatment of various diseases and currently represent the main source of a variety of phytocompounds, such as triterpenes. Pentacyclic triterpenes have been subjected to numerous studies that have revealed various biological activities, such as anticancer, antidiabetic, anti-inflammatory, antimicrobial, and hepatoprotective effects, which can be employed in therapy. However, due to their high lipophilicity, which is considered to exert a significant influence on their bioavailability, their current use is limited. A frequent approach employed to overcome this obstacle is the chemical derivatization of the core structure with different types of moieties including heterocycles, which are considered key elements in medicinal chemistry. The present review aims to summarize the literature published in the last 10 years regarding the derivatives of pentacyclic triterpenes bearing heterocyclic moieties and focuses on the biologically active derivatives as well as their structure-activity relationships. Predominantly, the targeted positions for the derivatization of the triterpene skeleton are C-3 (hydroxyl/oxo group), C-28 (hydroxyl/carboxyl group), and C-30 (allylic group) or the extension of the main scaffold by fusing various heterocycles with the A-ring of the phytocompound. In addition, numerous derivatives also contain linker moieties that connect the triterpenic scaffold with heterocycles; one such linker, the triazole moiety, stands out as a key pharmacophore for its biological effect. All these studies support the hypothesis that triterpenoid conjugates with heterocyclic moieties may represent promising candidates for future clinical trials.

Miniaturized Chemical Tags for Optical Imaging.

Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms (e.g., single aryl fluorophores, Raman-active triple bonds and isotopes) and embed them into building blocks (e.g., amino acids, nucleobases, sugars) to construct native-like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging.

Solid-phase synthesis and evaluation of linear and cyclic ferrocenoyl/ruthenocenoyl water-soluble hexapeptides as potential antibacterial compounds.

A series of novel water-soluble short peptide-bioconjugates containing a ferrocenoyl (Fc) or ruthenocenoyl (Rc) unit was synthesized and characterized to combine the unique activity of ferrocene and the isoelectronic ruthenocene with precisely designed peptide structures. We aim at evaluating these bioconjugates as a new class of OrganoMetallic Short AntiMicrobial Peptides (OM-SAMPs). The series of OM-SAMPs was designed with a set of linear and "head-to-tail" cyclic metallocene-based hexapeptides derived from the homo-sequence H-KKKKKK-NH2 by substitution of lysine (K) by tryptophan (W) and by orthogonal derivatization of the ε-N-amine group of lysine by a metallocene moiety. Peptide conjugates were characterized by RP-HPLC, mass spectrometry (ESI and MALDI-TOF) and circular dichroism (CD) spectroscopy. Gram-positive and Gram-negative antibacterial activity testings were carried out to explore the role of insertion of the metallocene fragment into the peptide, and the effect of the modification of the cationic charge and aromatic residues on the physiochemical properties of these OM-SAMPs. These results show that the insertion of two tryptophan residues and ferrocenoyl/ruthenocenoyl moieties into a linear homo-sequence peptides increase significantly their antibacterial activity with minimum inhibitory concentration values as low as 5 µM for the most active compounds. However, "head-to-tail" cyclic metallocene-based hexapeptides were not active against Gram-negative bacteria up to concentrations of 50 µM. These studies provide a better understanding of the role of structural modifications to enhance antibacterial peptide activity, which is promising for their therapeutic application.

Synthesis and bioconjugation of alkanethiol-stabilized gold bipyramid nanoparticles.

Gold bipyramid (GBP) nanoparticles are promising for a range of biomedical applications, including biosensing and surface-enhanced Raman spectroscopy, due to their favorable optical properties and ease of chemical functionalization. Here we report improved synthesis methods, including preparation of gold seed particles with an increased shelf life of ∼1 month, and preparation of GBPs with significantly shortened synthesis time (< 1 h). We also report methods for the functionalization and bioconjugation of the GBPs, including functionalization with alkanethiol self-assembled monolayers (SAMs) and bioconjugation with proteins via carbodiimide cross-linking. Binding of specific antibodies to the nanoparticle-bound proteins was subsequently observed via localized surface plasmon resonance sensing. Rabbit IgG and goat anti-Rabbit IgG antibodies were used as a model system for antibody-antigen interactions. As-synthesized, SAM-functionalized, and bioconjugated bipyramids were characterized using scanning electron microscopy, UV-vis spectroscopy, zeta potential, and dynamic light scattering.

Evaluation of the Physico-Chemical Properties of Liposomes Assembled from Bioconjugates of Anisic Acid with Phosphatidylcholine.

The aim of this work was the evaluation of the physico-chemical properties of a new type of liposomes that are composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine. In particular, the impact of modified anisic acid phospholipids on the thermotropic parameters of liposomes was determined, which is crucial for using them as potential carriers of active substances in cancer therapies. Their properties were determined using three biophysical methods, namely differential scanning calorimetry (DSC), steady-state fluorimetry and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Moreover, temperature studies of liposomes composed of DPPC and bioconjugates of anisic acid with phosphatidylcholine provided information about the phase transition, fluidity regarding chain order, hydration and dynamics. The DSC results show that the main phase transition peak for conjugates of anisic acid with phosphatidylcholine molecules was broadened and shifted to a lower temperature in a concentration- and structure-dependent manner. The ATR-FTIR results and the results of measurements conducted using fluorescent probes located at different regions in the lipid bilayer are in line with DSC. The results show that the new bioconjugates with phosphatidylcholine have a significant impact on the physico-chemical properties of a membrane and cause a decrease in the temperature of the main phase transition. The consequence of this is greater fluidity of the lipid bilayer.

Structural Characterization of Daunomycin-Peptide Conjugates by Various Tandem Mass Spectrometric Techniques.

The use of peptide-drug conjugates has generated wide interest as targeted antitumor therapeutics. The anthracycline antibiotic, daunomycin, is a widely used anticancer agent and it is often conjugated to different tumor homing peptides. However, comprehensive analytical characterization of these conjugates via tandem mass spectrometry (MS/MS) is challenging due to the lability of the O-glycosidic bond and the appearance of MS/MS fragment ions with little structural information. Therefore, we aimed to investigate the optimal fragmentation conditions that suppress the prevalent dissociation of the anthracycline drug and provide good sequence coverage. In this study, we comprehensively compared the performance of common fragmentation techniques, such as higher energy collisional dissociation (HCD), electron transfer dissociation (ETD), electron-transfer higher energy collisional dissociation (EThcD) and matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) activation methods for the structural identification of synthetic daunomycin-peptide conjugates by high-resolution tandem mass spectrometry. Our results showed that peptide backbone fragmentation was inhibited by applying electron-based dissociation methods to conjugates, most possibly due to the "electron predator" effect of the daunomycin. We found that efficient HCD fragmentation was largely influenced by several factors, such as amino acid sequences, charge states and HCD energy. High energy HCD and MALDI-TOF/TOF combined with collision induced dissociation (CID) mode are the methods of choice to unambiguously assign the sequence, localize different conjugation sites and differentiate conjugate isomers.