Evaluating the In Vitro Activity and Safety of Modified LfcinB Peptides as Potential Colon Anticancer Agents: Cell Line Studies and Insect-Based Toxicity Assessments.

Anticancer peptides are increasingly being considered as alternative treatments for cancer due to their potency, selectivity, and low toxicity. Previously, the peptide LfcinB (21-25)Pal showed in vitro anticancer effects against the Caco-2 colon cancer cell line (half-maximal inhibitory concentration (IC50): 86 µM). In this study, we developed modifications to the peptide sequence to increase its anticancer activity. Sequence modifications were made such as the inclusion of amino hexanoic acid (Ahx), N-terminal biotinylation, acetylation, and substitutions of Orn for Arg and/or d-Arg by l-Arg. The molecules were synthesized using manual solid-phase peptide synthesis (SPPS), and their synthetic feasibility (SAScore) ranged from 6.2 to 7.6. The chromatographic purities of the synthesized peptides were greater than 89%. We found that Ahx-RWQWRWQWR and RWQWRWQW-Orn showed activity against both Caco-2 and HT-29 cell lines and decreased IC50 values by approx. 50% in Caco-2 cells (IC50: 40 µM) when compared to the parent peptide RWQWRWQWR. Moreover, the modified peptides demonstrated lower hemolytic effects, with values <10% at 200 µg/mL. Toxicity was assessed using the Galleria mellonella model and the half-maximal lethal dose (LD50) for the best peptides was >100 mg/kg, indicating that their toxicity is classified as moderately toxic or lower. In contrast, cisplatin showed an LD50 of 13 mg/Kg. The designed anticancer peptides presented good in vitro activity and low toxicity, making them promising molecules for future drug development studies.

In-house standards derived from doping peptides: Enzymatic and serum stability and degradation profile of GHRP and GHRH-related peptides.

Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide (26 [F] LfcinB (20-30)2 ) in four different matrices to test these peptides as in-house IS.

Non-natural amino acids into LfcinB-derived peptides: effect in their (i) proteolytic degradation and (ii) cytotoxic activity against cancer cells.

The dimeric peptide 26[F]: (RRWQWRFKKLG)2-K-Ahx has exhibited a potent cytotoxic effect against breast cancer cell lines, with position 26 (F) being the most relevant for anti-cancer activity. In this investigation, six analogues of the 26[F] peptide were synthesized in which the 26th position was replaced by non-natural hydrophobic amino acids, finding that some modifications increased the resistance to proteolytic degradation exerted by trypsin or pepsin. Additionally, these modifications increased the cytotoxic effect against breast cancer cells and generated cell death mediated by apoptosis pathways, activating caspases 8 and 9, and did not compromise the integrity of the cytoplasmic membrane. Finally, it was found that the modified peptides have a broad spectrum of action, since they also have a cytotoxic effect against the HeLa human cervical cancer cell line. Peptide 26[F] was inoculated in mice by ip administration and the lethal dose 50 (LD50) was between 70 and 140 mg kg-1. While for the 26[1-Nal]: (RRWQWR-1-Nal-KKLG)2-K-Ahx peptide, a dose-response test was performed, and the survival rate was 100%. These results suggested that these peptides are safe in this animal model and could be considered as promissory to develop a treatment against breast cancer.

Peptide-Resorcinarene Conjugates Obtained via Click Chemistry: Synthesis and Antimicrobial Activity.

Antimicrobial resistance (AMR) is one of the top ten threats to public health, as reported by the World Health Organization (WHO). One of the causes of the growing AMR problem is the lack of new therapies and/or treatment agents; consequently, many infectious diseases could become uncontrollable. The need to discover new antimicrobial agents that are alternatives to the existing ones and that allow mitigating this problem has increased, due to the rapid and global expansion of AMR. Within this context, both antimicrobial peptides (AMPs) and cyclic macromolecules, such as resorcinarenes, have been proposed as alternatives to combat AMR. Resorcinarenes present multiple copies of antibacterial compounds in their structure. These conjugate molecules have exhibited antifungal and antibacterial properties and have also been used in anti-inflammatory, antineoplastic, and cardiovascular therapies, as well as being useful in drug and gene delivery systems. In this study, it was proposed to obtain conjugates that contain four copies of AMP sequences over a resorcinarene core. Specifically, obtaining (peptide)4-resorcinarene conjugates derived from LfcinB (20-25): RRWQWR and BF (32-34): RLLR was explored. First, the synthesis routes that allowed obtaining: (a) alkynyl-resorcinarenes and (b) peptides functionalized with the azide group were established. These precursors were used to generate (c) (peptide)4-resorcinarene conjugates by azide-alkyne cycloaddition CuAAC, a kind of click chemistry. Finally, the conjugates' biological activity was evaluated: antimicrobial activity against reference strains and clinical isolates of bacteria and fungi, and the cytotoxic activity over erythrocytes, fibroblast, MCF-7, and HeLa cell lines. Our results allowed establishing a new synthetic route, based on click chemistry, for obtaining macromolecules derived from resorcinarenes functionalized with peptides. Moreover, it was possible to identify promising antimicrobial chimeric molecules that may lead to advances in the development of new therapeutic agents.

Changes in Length and Positive Charge of Palindromic Sequence RWQWRWQWR Enhance Cytotoxic Activity against Breast Cancer Cell Lines.

Breast cancer is one of the main causes of premature death in women; current treatments have low selectivity, generating strong physical and psychological sequelae. The palindromic peptide R-1-R (RWQWRWQWR) has cytotoxic activity against different cell lines derived from cancer and selectivity against noncancerous cells. To determine if changes in the charge/length of this peptide increase its activity, six peptides were obtained by SPPS, three of them with addition of Arg at the N, C-terminal or both and three with deletion of Arg at the N, C-terminal or both. The cytotoxic and selective activities were evaluated against MCF-7, MDA-MB-231, and MCF-12 cell lines and fibroblast primary cell culture, evidencing that the RR-1-R peptide with the inclusion of Arg in the N-terminal end maintained selectivity and increased cytotoxicity against lines derived from breast cancer. The effect of this addition regarding the type of induced cell death was evaluated by flow cytometry, showing very low rates of necrosis and a significant majority of apoptotic events with activation of both Caspase 8 and Caspase 9. This work allowed us to find a modification that generates a peptide with greater cytotoxic effects and can be considered a promising molecule for other approaches to improve anticancer peptides.

Gradient Retention Factor Concept Applied to Method Development for Peptide Analysis by Means of RP-HPLC.

Using the van Deemter model, the efficiency of three stationary phase systems in the analysis of a mixture of synthetic peptides was evaluated: (i) monolithic, (ii) packed, and (iii) core-shell columns, and it was shown that the efficiency of the monolithic column is superior to the others, specifically using it, the lowest values of H min (0.03 and 0.1 mm) were obtained, and additionally its efficiency was not significantly affected by increasing the flow. Using the concept of the gradient retention factor (k*), a method for chromatographic separation of a peptide complex mixture was designed, implemented, and optimized and then transferred from a packed column to a monolithic one. The results showed that it was possible to separate all components of the mixture using both evaluated columns; moreover, the analysis time was reduced from 70 to 10 min, conserving the critical pair resolution (1.4), by the transfer method using the k* concept. The method developed was tested against a mixture of doping peptides, showing that this method is efficient for separating peptides of various natures. This investigation is very useful for the development of methods for the analysis of complex peptide mixtures since it provides a systematic approach that can be extrapolated to different types of columns and instrumentation.

Peptides Derived from (RRWQWRMKKLG)2-K-Ahx Induce Selective Cellular Death in Breast Cancer Cell Lines through Apoptotic Pathway.

The effect on the cytotoxicity against breast cancer cell lines of the substitution of 26Met residue in the sequence of the Bovine Lactoferricin-derived dimeric peptide LfcinB (20-30)2: (20RRWQWRMKKLG30)2-K-Ahx with amino acids of different polarity was evaluated. The process of the synthesis of the LfcinB (20-30)2 analog peptides was similar to the original peptide. The cytotoxic assays showed that some analog peptides exhibited a significant cytotoxic effect against breast cancer cell lines HTB-132 and MCF-7, suggesting that the substitution of the Met with amino acids of a hydrophobic nature drastically enhances its cytotoxicity against HTB-132 and MCF-7 cells, reaching IC50 values up to 6 µM. In addition, these peptides have a selective effect, since they exhibit a lower cytotoxic effect on the non-tumorigenic cell line MCF-12. Interestingly, the cytotoxic effect is fast (90 min) and is maintained for up to 48 h. Additionally, through flow cytometry, it was found that the obtained dimeric peptides generate cell death through the apoptosis pathway and do not compromise the integrity of the cytoplasmic membrane, and there are intrinsic apoptotic events involved. These results show that the obtained peptides are extremely promising molecules for the future development of drugs for use against breast cancer.

Short peptides conjugated to non-peptidic motifs exhibit antibacterial activity.

Short peptides derived from buforin and lactoferricin B were conjugated with other antimicrobial molecules of different chemical natures. The sequences RLLR, RLLRLLR, RWQWRWQWR, and RRWQWR were conjugated at their N-terminal end with non-peptidic molecules such as 6-aminohexanoic acid, ferrocene, caffeic acid, ferulic acid, and oxolinic acid. Peptide conjugates and unmodified peptides were synthesized by means of solid-phase peptide synthesis using the Fmoc/tBu strategy (SPPS-Fmoc/tBu), purified via RP-SPE, and characterized via RP-HPLC and MS. The peptides' antibacterial activity against bacterial strains E. coli ATCC 25922 and S. aureus ATCC 25923 was evaluated, and the results showed that the peptide conjugates exhibited higher antibacterial activity than the original unconjugated peptides. Conjugation of AMPs is a promising strategy for designing and identifying new drugs for treating bacterial infections.

Selective cytotoxic effect against the MDA-MB-468 breast cancer cell line of the antibacterial palindromic peptide derived from bovine lactoferricin.

The cytotoxic effect against the breast cancer cell line MDA-MB-468 of the palindromic peptide LfcinB (21-25)Pal: 1RWQWRWQWR9 and its analogous peptides, obtained via alanine scanning, was evaluated. The results indicate that the palindromic peptide exhibited a concentration-dependent cytotoxic effect against this cell line. The cytotoxic effect of the palindromic peptide was fast and selective and was sustained for up to 48 h of treatment. MDA-MB-468 cells treated with the palindromic peptide exhibited severe cellular damage, acquiring rounded forms and shrinkage, a behavior typical of apoptotic events. The analogous peptides exhibited fewer cytotoxic effects than the original palindromic peptide, suggesting that the substitution of any amino acid with alanine diminishes the cytotoxic effect. The Arg and Trp residues proved to be the most relevant for the cytotoxic effect; the analogous peptides with substitutions of Trp with Ala did not induce a change in cellular morphology, while analogous peptides with substitutions of Arg or Gln with Ala induced cellular damage. Also, neither the palindromic peptide nor its analogues exerted a significant cytotoxic effect on normal fibroblasts, indicating that the peptides had a selective cytotoxic effect on cancerous cells. The peptide LfcinB (21-25)Pal, and its analogues exhibited antibacterial activity against E. coli and S. aureus strains and a selective cytotoxic effect against the breast cancer cell line MDA-MB-468.

Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology.

A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.

Pullulan nanofibers containing the antimicrobial palindromic peptide LfcinB (21-25)Pal obtained via electrospinning.

Electrospinning technology is useful for making ultrafine drug-eluting fibers for the clinical treatment of wounds. We show the incorporation of an antimicrobial LfcinB-derived peptide into Pullulan nanofibers. The palindromic peptide LfcinB (21-25)Pal: RWQWRWQWR was synthesized, purified, and characterized by means of the RP-HPLC and MALDI-TOF MS methods. The peptide's antibacterial activity against the E. coli ATCC 25922 strain was evaluated, and the peptide LfcinB (20-25)Pal exhibited significant antibacterial activity. Nanofibers were obtained by electrospinning a Pullulan or Pullulan-LfcinB (21-25)Pal solution. The obtained nanofibers were characterized via microscopy (AFM and SEM) and RP-HPLC chromatography. The peptide incorporation efficiency was 31%. The Pullulan-LfcinB (21-25)Pal nanofibers were soluble in water, and the peptide was liberated immediately. The Pullulan-LfcinB (21-25)Pal nanofibers exhibited the same antibacterial activity against E. coli strain as the free peptide LfcinB (21-25)Pal. The results suggest that Pullulan-LfcinB (21-25)Pal nanofibers could be considered for designing and developing antibacterial wound dressings.

The tetrameric peptide LfcinB (20-25)4 derived from bovine lactoferricin induces apoptosis in the MCF-7 breast cancer cell line.

The cytotoxic effect of the tetrameric peptide LfcinB (20-25)4 against breast cancer cell line ATCC® HTB-22™ (MCF-7) was evaluated. The tetrameric peptide exhibited a concentration-dependent cytotoxic effect against MCF-7 cancer cells. The peptide at 22 µM had the maximum cytotoxic effect against MCF-7 cancer cells, reducing their cell viability to ∼20%. The cytotoxic effect of the tetrameric peptide against MCF-7 cells was sustained for 24 hours. Furthermore, the tetrameric peptide did not exhibit a significant cytotoxic effect against the non-tumorogenic trophoblastic cell line, which confirms their selectivity for breast cancer cell lines. The MCF-7 cells treated at 12.2 µM for 1 h exhibited morphological changes characteristic of apoptosis, such as rounded forms and cellular shrinkage. Furthermore, this peptide induces severe cellular damage to MCF-7 cells, mitochondrial membrane depolarization, and increase of cytoplasmic calcium concentration. Our results suggest that it has a significant selective cytotoxic effect against MCF-7 cells, which may be mainly associated with the apoptotic pathway. This peptide, which contains the RRWQWR motif, could be considered to be a promising candidate for developing therapeutic agents for the treatment of breast cancer.

Synergistic bactericide and antibiotic effects of dimeric, tetrameric, or palindromic peptides containing the RWQWR motif against Gram-positive and Gram-negative strains.

Dimeric and tetrameric peptides derived from LfcinB (20-25): RRWQWR, LfcinB (20-30): RRWQWRMKKLG, LfcinB (17-31): FKARRWQWRMKKLGA, or the palindromic sequence LfcinB (21-25)Pal: RWQWRWQWR were obtained by means of the SPPS-Fmoc/tBu methodology. The antibacterial activity of these molecules was evaluated against Escherichia coli (ATCC 25922 and ATCC 11775), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), and Pseudomonas aeruginosa (ATCC 27853). The dimer LfcinB (20-25)2: (RRWQWR)2K-Ahx, the tetramer LfcinB (20-25)4: (RRWQWR)4K2-Ahx2-C2, and the palindromic sequence LfcinB (21-25)Pal exhibited the highest antibacterial activity against the tested bacterial strains. In all cases, the antibacterial activity was dependent on peptide concentration. The polyvalent molecules LfcinB (20-25)2 and LfcinB (20-25)4 exhibited bacteriostatic and bactericidal activity against E. coli, P. aeruginosa, and S. aureus strains; additionally, this dimer and this tetramer combined with ciprofloxacin exhibited a synergistic antibacterial effect against E. coli ATCC 25922 and P. aeruginosa, respectively. Furthermore, the peptides LfcinB (20-30)4, LfcinB (20-25)4, and LfcinB (21-25)Pal combined with vancomycin exhibited a synergistic antibacterial effect against S. aureus and E. faecalis, respectively. This study showed that polyvalent peptides derived from LfcinB exhibit significant antibacterial activity, suggesting that these peptides could have a therapeutic application. Furthermore, our results suggest that polyvalent peptide synthesis could be considered as an innovative and viable strategy for obtaining promising antimicrobial molecules.

Design, Synthesis, and Use of Peptides Derived from Human Papillomavirus L1 Protein for the Modification of Gold Electrode Surfaces by Self-Assembled Monolayers.

In order to obtain gold electrode surfaces modified with Human Papillomavirus L1 protein (HPV L1)-derived peptides, two sequences, SPINNTKPHEAR and YIK, were chosen. Both have been recognized by means of sera from patients infected with HPV. The molecules, Fc-Ahx-SPINNTKPHEAR, Ac-C-Ahx-(Fc)KSPINNTKPHEAR, Ac-C-Ahx-SPINNTKPHEAR(Fc)K, C-Ahx-SPINNTKPHEAR, and (YIK)2-Ahx-C, were designed, synthesized, and characterized. Our results suggest that peptides derived from the SPINNTKPHEAR sequence, containing ferrocene and cysteine residues, are not stable and not adequate for electrode surface modification. The surface of polycrystalline gold electrodes was modified with the peptides C-Ahx-SPINNTKPHEAR or (YIK)2-Ahx-C through self-assembly. The modified polycrystalline gold electrodes were characterized via infrared spectroscopy and electrochemical measurements. The thermodynamic parameters, surface coverage factor, and medium pH effect were determined for these surfaces. The results indicate that surface modification depends on the peptide sequence (length, amino acid composition, polyvalence, etc.). The influence of antipeptide antibodies on the voltammetric response of the modified electrode was evaluated by comparing results obtained with pre-immune and post-immune serum samples.

Adhesión convencional en dentina, dificultades y avances en la técnica / Conventional dentin bonding. Difficulties and progress in the technique

INTRODUCCIÓN: estudios respecto a la adhesión en dentina han reportado que, contrario a la estabilidad lograda sobre esmalte dental, en dentina los mecanismos adhesivos todavía son sensibles, impredecibles e inestables. El objetivo de este trabajo es revisar la literatura actual sobre la adhesión en dentina, con el fin de caracterizar la adhesión convencional describiendo las modificaciones actuales del protocolo convencional, encaminadas a mejorar el desempeño adhesivo de los materiales dentales. Métodos: se hizo una revisión de la literatura evaluando 3 bases de datos: ScienceDirect, Springer y Medline, de las cuales se escogieron los 52 artículos más relevantes, publicados entre los años 2004 y 2013. Se usaron, como criterios de búsqueda, las palabras clave: dentin, dentin bonding, bond strength y acid etching. Resultados: al revisar los artículos seleccionados, se logró una descripción del protocolo de adhesión convencional que muestra la formación del barrillo dentinario (smear layer), la acción del ácido fosfórico y la formación de la interfase adhesiva propiamente dicha, junto con las dificultades propias de la técnica y las posibles soluciones planteadas hasta la fecha. Conclusión: la adhesión convencional sobre dentina es un procedimiento estricto y delicado, que evidencia inconvenientes como la degradación hidrolítica y proteolítica de la matriz de colágeno por parte de enzimas liberadas en el momento de la desmineralización, lo que deteriora la interfase adhesiva. Por tanto, se han sugerido sustancias que pueden ser utilizadas como agentes de protección del colágeno, sin alterar e incluso mejorando la resistencia adhesiva.

INTRODUCTION: studies on dentin bonding have reported that, contrary to the achieved stability on dental enamel, adhesive mechanisms on dentine are still sensitive, unpredictable, and unstable. The objective of this study is to review the current literature on dentin bonding in order to characterize conventional bonding, describing current modifications of the conventional protocol aimed at improving the adhesive performance of dental materials. METHODS: a literature review was conducted within 3 databases: ScienceDirect, Springer, and Medline, choosing the 52 most relevant articles published between 2004 and 2013. The following key words were used as search criteria: dentin, dentin bonding, bond strength, and acid etching. RESULTS: the review of the selected articles provided a description of the conventional adhesion protocol showing the formation of smear layer, the action of phosphoric acid, and the actual formation of adhesive interface, as well as the difficulties of the technique and possible solutions suggested to date. CONCLUSIONS: conventional dentin bonding is a precise and delicate procedure that shows disadvantages such as the hydrolytic and proteolytic degradation of collagen matrix by enzymes released at the time of demineralization, which damages the adhesive interface. Therefore, several substances have been suggested to be used as agents of collagen protection without altering adhesive strength, and even improving it.studies on dentin bonding have reported that, contrary to the achieved stability on dental enamel, adhesive mechanisms on dentine are still sensitive, unpredictable, and unstable. The objective of this study is to review the current literature on dentin bonding in order to characterize conventional bonding, describing current modifications of the conventional protocol aimed at improving the adhesive performance of dental materials. Methods: a literature review was conducted within 3 databases: ScienceDirect, Springer, and Medline, choosing the 52 most relevant articles published between 2004 and 2013. The following key words were used as search criteria: dentin, dentin bonding, bond strength, and acid etching. Results: the review of the selected articles provided a description of the conventional adhesion protocol showing the formation of smear layer, the action of phosphoric acid, and the actual formation of adhesive interface, as well as the difficulties of the technique and possible solutions suggested to date. Conclusion: conventional dentin bonding is a precise and delicate procedure that shows disadvantages such as the hydrolytic and proteolytic degradation of collagen matrix by enzymes released at the time of demineralization, which damages the adhesive interface. Therefore, several substances have been suggested to be used as agents of collagen protection without altering adhesive strength, and even improving it.