Harnessing exosomes as a platform for drug delivery in breast cancer: A systematic review for in vivo and in vitro studies.

Breast cancer remains a significant global health concern, emphasizing the critical need for effective treatment strategies, especially targeted therapies. This systematic review summarizes the findings from in vitro and in vivo studies regarding the therapeutic potential of exosomes as drug delivery platforms in the field of breast cancer treatment. A comprehensive search was conducted across bibliographic datasets, including Web of Science, PubMed, and Scopus, using relevant queries from several related published articles and the Medical Subject Headings Database. Then, all morphological, biomechanical, histopathological, and cellular-molecular outcomes were systematically collected. A total of 30 studies were identified based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. These studies underwent assessment using the Systematic Review Centre for Laboratory Animal Experimentation risk of bias assessment tool. The results indicate that exosomes exhibit promise as effective drug delivery platforms, capable of hindering cancer cell viability, proliferation, migration, and angiogenesis. However, a comprehensive assessment is challenging due to some studies deviating from guidelines and having incomplete methodology. Addressing these, future studies should detail methodologies, optimize dosing, and enhance exosome production. Standardization in reporting, consistent protocols, and exploration of alternative sources are crucial.

Proteomic characterization and cytotoxic potential of proteins from Cuscuta (Cuscuta epithymum (L.) crude herbal product against MCF-7 human breast cancer cell line.

BACKGROUND: The burden of breast cancer, the second leading cause of death worldwide, is increasing at an alarming rate. Cuscuta, used in traditional medicine for different ailments, including cancer, is known for containing phytochemicals that exhibit anticancer activity; however, the bioactivities of proteins from this plant remain unexplored. This study aimed to screen the cytotoxic potential of proteins from the crude herbal product of Cuscuta epithymum(L.) (CE) harvested from the host plants Alhagi maurorum and Medicago sativa. METHODS: The proteins from CE were extracted using a salting-out method, followed by fractionation with a gel filtration chromatography column. Gel-free shotgun proteomics was subsequently performed for protein characterization. The viability assay using MTT was applied to deduce the cytotoxic potential of proteins against MCF-7 breast cancer cells, with further exploration of the effect of treatment on the expression of the apoptotic mediator BCL2-associated X protein (BAX) and B-cell lymphoma protein 2 (BCL-2) proteins, using western blotting to strengthen the findings from the in vitro viability assay. RESULTS: The crude proteins (CP) of CE were separated into four protein peaks (P1, P2, P3, and P4) by gel filtration chromatography. The evaluation of potency showed a dose-dependent decline in the MCF-7 cell line after CP, P1, P2, and P3 treatment with the respective IC50 values of 33.8, 43.1, 34.5, and 28.6 µg/ml. The percent viability of the cells decreased significantly upon treatment with 50 µg/ml CP, P1, P2, and P3 (P < 0.001). Western-blot analysis revealed upregulation of proapoptotic protein BAX in the cells treated with CP, P3 (P < 0.01), and P2 (P < 0.05); however, the antiapoptotic protein, BCL-2 was downregulated in the cells treated with CP and P3 (P < 0.01), but no significant change was detected in P2 treated cells. The observed cytotoxic effects of proteins in the CP, P1, P2, and P3 from the in vitro viability assay and western blot depicted the bioactivity potential of CE proteins. The database search revealed the identities of functionally important proteins, including nonspecific lipid transfer protein, superoxide dismutase, carboxypeptidase, RNase H domain containing protein, and polyribonucleotide nucleotidyltransferase, which have been previously reported from other plants to exhibit anticancer activity. CONCLUSION: This study indicated the cytotoxic activity of Cuscuta proteins against breast cancer MCF-7 cells and will be utilized for future investigations on the mechanistic effect of active proteins. The survey of CE proteins provided substantial data to encourage further exploration of biological activities exhibited by proteins in Cuscuta.

Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery.

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

Design, Synthesis, and Antiproliferative Activity of Benzopyran-4-One-Isoxazole Hybrid Compounds.

The biological significance of benzopyran-4-ones as cytotoxic agents against multi-drug resistant cancer cell lines and isoxazoles as anti-inflammatory agents in cellular assays prompted us to design and synthesize their hybrid compounds and explore their antiproliferative activity against a panel of six cancer cell lines and two normal cell lines. Compounds 5a-d displayed significant antiproliferative activities against all the cancer cell lines tested, and IC50 values were in the range of 5.2-22.2 µM against MDA-MB-231 cancer cells, while they were minimally cytotoxic to the HEK-293 and LLC-PK1 normal cell lines. The IC50 values of 5a-d against normal HEK-293 cells were in the range of 102.4-293.2 µM. Compound 5a was screened for kinase inhibitory activity, proteolytic human serum stability, and apoptotic activity. The compound was found inactive towards different kinases, while it completely degraded after 2 h of incubation with human serum. At 5 µM concentration, it induced apoptosis in MDA-MB-231 by 50.8%. Overall, these findings suggest that new benzopyran-4-one-isoxazole hybrid compounds, particularly 5a-d, are selective anticancer agents, potentially safe for human cells, and could be synthesized at low cost. Additionally, Compound 5a exhibits potential anticancer activity mediated via inhibition of cancer cell proliferation and induction of apoptosis.

Amphiphilic Cell-Penetrating Peptides Containing Arginine and Hydrophobic Residues as Protein Delivery Agents.

The entry of proteins through the cell membrane is challenging, thus limiting their use as potential therapeutics. Seven cell-penetrating peptides, designed in our laboratory, were evaluated for the delivery of proteins. Fmoc solid-phase peptide synthesis was utilized for the synthesis of seven cyclic or hybrid cyclic-linear amphiphilic peptides composed of hydrophobic (tryptophan (W) or 3,3-diphenylalanine (Dip) and positively-charged arginine (R) residues, such as [WR]4, [WR]9, [WWRR]4, [WWRR]5, [(RW)5K](RW)5, [R5K]W7, and [DipR]5. Confocal microscopy was used to screen the peptides as a protein delivery system of model cargo proteins, green and red fluorescein proteins (GFP and RFP). Based on the confocal microscopy results, [WR]9 and [DipR]5 were found to be more efficient among all the peptides and were selected for further studies. [WR]9 (1-10 µM) + protein (GFP and RFP) physical mixture did not show high cytotoxicity (>90% viability) in triple-negative breast cancer cells (MDA-MB-231) after 24 h, while [DipR]5 (1-10 µM) physical mixture with GFP exhibited more than 81% cell viability. Confocal microscopy images revealed internalization of GFP and RFP in MDA-MB-231 cells using [WR]9 (2-10 µM) and [DipR]5 (1-10 µM). Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptake of GFP was concentration-dependent in the presence of [WR]9 in MDA-MB-231 cells after 3 h of incubation at 37 °C. The concentration-dependent uptake of GFP and RFP was also observed in the presence of [DipR5] in SK-OV-3 and MDA-MB-231 cells after 3 h of incubation at 37 °C. FACS analysis indicated that the cellular uptake of GFP in the presence of [WR]9 was partially decreased by methyl-ß-cyclodextrin and nystatin as endocytosis inhibitors after 3 h of incubation in MDA-MB-231 cells, whereas nystatin and chlorpromazine as endocytosis inhibitors slightly reduced the uptake of GFP in the presence of [DipR]5 after 3 h of incubation in MDA-MB-231. [WR]9 was able to deliver therapeutically relevant proteins (Histone H2A) at different concentrations. These results provide insight into the use of amphiphilic cyclic peptides in the delivery of protein-related therapeutics.

Modified Linear Peptides Effectively Silence STAT-3 in Breast Cancer and Ovarian Cancer Cell Lines.

RNA interference (RNAi) has drawn enormous attention as a powerful tool because of its capability to interfere with mRNA and protein production. However, designing a safe and efficient delivery system in RNAi therapeutics remains challenging. Herein, we have designed and synthesized several linear peptides containing tryptophan (W) and arginine (R) residues separated by the ß-alanine (ßA) spacer and attached to a lipophilic fatty acyl chain, cholesterol, or PEG. The peptide backbone sequences were: Ac-C-ßA-ßA-W4-ßA-ßA-R4-CO-NH2 and Ac-K-ßA-ßA-W4-ßA-ßA-R4-CO-NH2, with only a difference in N-terminal amino acid. The cysteine side chain in the first sequence was used for the conjugation with PEG2000 and PEG550. Alternatively, the side chain of lysine in the second sequence was used for conjugation with cholesterol or oleic acid. We hypothesized that amphiphilic peptides and optimum fatty acyl chain or PEG could function as an effective siRNA carrier by complementing each structural component's self-assembly and membrane internalization properties. None of the designed peptides showed cytotoxicity up to 10 µM. Serum stability studies suggested that the newly designed peptides efficiently protected siRNA against early degradation by nucleases. Flow cytometry analysis indicated 50-90% cellular uptake of siRNA using the newly developed modified linear peptides (MLPs). Western blot results revealed more than 90% protein downregulation after targeting STAT3 in MDA-MB-231 and SKOV-3 cell lines. In summary, a new peptide class was developed to safely and efficiently deliver siRNA.

Cyclic and Linear Peptides Containing Alternate WW and RR Residues as Molecular Cargo Delivery Tools.

Cell-impermeable and negatively charged compounds' cellular uptake across the cell membranes remains challenging. Herein, the synthesis of four linear [(WWRR)2, (WWRR)3, (WWRR)4, and (WWRR)5] and four cyclic ([WWRR]2, [WWRR]3, [WWRR]4, and [WWRR]5) peptides containing alternate two tryptophan (WW) and two arginine (RR) residues and their biological evaluation as molecular transporters are reported. The peptides did not show any significant cytotoxicity in different cell lines (MDA-MB-23, SK-OV-3, and HEK 293) at a concentration of 5 µM and after 3 h of incubation time. The uptake of fluorescence-labeled cargo molecules (F'-GpYEEI, F'-siRNA, and F'-3TC) in the presence of the peptides was monitored in different cell lines (SK-OV-3 and MDA-MB-231) with fluorescence-activated cell sorting. Among all the peptides, [WWRR]5 (C4) showed the highest cellular uptake of cargo molecules, indicating it can act as effective molecular transporter. Confocal microscopy in MDA-MB-231 cells showed the cellular uptake of F'-GpYEEI in the presence of C4 and the intracellular localization of fluorescence-labeled C4 (F'-C4) in the cytosol. The F'-C4 cellular uptake was found to be concentration- and time-dependent, as shown by flow cytometry in MDA-MB-231 cells. Confocal microscopy and flow cytometry of F'-C4 in MDA-MB-231 cells were examined alone and in the presence of different endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and nystatin). The data showed that the cellular uptake of F'-C4 in the presence of chlorpromazine, chloroquine, and methyl-ß-cyclodextrin was reduced but not completely eliminated, indicating that both energy-independent and energy-dependent pathways contributed to the cellular uptake of F'-C4. Similar results were obtained using the confocal microscopy of C4 and F'-GpYEEI in the presence of endocytosis inhibitors (chlorpromazine, methyl-ß-cyclodextrin, chloroquine, and nystatin). These data indicate that C4 has the potential to be used as a cell-penetrating peptide and cargo transporter.

Bis-Cinnamamide Derivatives as APE/Ref-1 Inhibitors for the Treatment of Human Melanoma.

Human malignant melanoma exhibits imbalances in redox status, leading to activation of many redox-sensitive signaling pathways. APE/Ref-1 is a multifunctional protein that serves as a redox chaperone that regulates many nuclear transcription factors and is an important mechanism in cancer cell survival of oxidative stress. Previous studies showed that APE/Ref-1 is a potential druggable target for melanoma therapy. In this study, we synthesized a novel APE/Ref-1 inhibitor, bis-cinnamoyl-1,12-dodecamethylenediamine (2). In a xenograft mouse model, compound 2 treatment (5 mg/kg) significantly inhibited tumor growth compared to the control group, with no significant systemic toxicity observed. We further synthesized compound 2 analogs to determine the structure-activity relationship based on their anti-melanoma activities. Among those, 4-hydroxyphenyl derivative (11) exhibited potent anti-melanoma activities and improved water solubility compared to its parental compound 2. The IC50 of compound 11 was found to be less than 0.1 µM. Compared to other known APE/Ref-1 inhibitors, compound 11 exhibited increased potency in inhibiting melanoma proliferation. As determined by luciferase reporter analyses, compound 2 was shown to effectively inhibit H2O2-activated AP-1 transcription activities. Targeting APE/Ref-1-mediated signaling using pharmaceutical inhibitors is a novel and effective strategy for melanoma treatment with potentially high impact.

Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents.

A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5-10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F'-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F'-[K(DipR)5]) alone and F'-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F'-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F'-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl ß-cyclodextrin, did not completely inhibit the cellular uptake of F'-[K(DipR)5] alone or F'-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

Redox-Responsive Disulfide Cyclic Peptides: A New Strategy for siRNA Delivery.

RNA interference (RNAi) is a powerful tool capable of targeting virtually any protein without time-consuming and expensive drug development studies. However, due to obstacles facing efficient and safe delivery, RNAi-based therapeutic approach remains a challenge. Herein, we have designed and synthesized a number of disulfide-constraining cyclic and hybrid peptides using tryptophan and arginine residues. Our hypothesis was that peptide structures would undergo reduction by intracellular glutathione (more abundant in cancer cells) and unpack the small interfering RNA (siRNA) from the peptide/siRNA complexes. A subset of newly developed peptides (specifically, C4 and H4) exhibited effective cellular internalization of siRNA (∼70% of the cell population; monitored by flow cytometry and confocal microscopy), the capability of protecting siRNA against early degradation by nucleases (monitored by gel electrophoresis), minimal cytotoxicity in selected cell lines (studied by cell viability and LC50 calculations), and efficient protein silencing by 70-75% reduction in the expression of targeting signal transducer and activator of transcription 3 (STAT3) in human triple-negative breast cancer (TNBC) MDA-MB-231 cells, analyzed using the Western blot technique. Our results indicate the birth of a promising new family of siRNA delivery systems that are capable of safe and efficient delivery, even in the presence of nucleases.

[(WR)8WKßA]-Doxorubicin Conjugate: A Delivery System to Overcome Multi-Drug Resistance against Doxorubicin.

Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR)8WKßA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR)8WKßA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 µM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 µM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR)8WKßA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 µM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t1/2) for [(WR)8WKßA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it.

A PDK-1 allosteric agonist neutralizes insulin signaling derangements and beta-amyloid toxicity in neuronal cells and in vitro.

The Alzheimer's brain is affected by multiple pathophysiological processes, which include a unique, organ-specific form of insulin resistance that begins early in its course. An additional complexity arises from the four-fold risk of Alzheimer's Disease (AD) in type 2 diabetics, however there is no definitive proof of causation. Several strategies to improve brain insulin signaling have been proposed and some have been clinically tested. We report findings on a small allosteric molecule that reverses several indices of insulin insensitivity in both cell culture and in vitro models of AD that emphasize the intracellular accumulation of ß-amyloid (Aßi). PS48, a chlorophenyl pentenoic acid, is an allosteric activator of PDK-1, which is an Akt-kinase in the insulin/PI3K pathway. PS48 was active at 10 nM to 1 µM in restoring normal insulin-dependent Akt activation and in mitigating Aßi peptide toxicity. Synaptic plasticity (LTP) in prefrontal cortical slices from normal rat exposed to Aß oligomers also benefited from PS48. During these experiments, neither overstimulation of PI3K/Akt signaling nor toxic effects on cells was observed. Another neurotoxicity model producing insulin insensitivity, utilizing palmitic acid, also responded to PS48 treatment, thus validating the target and indicating that its therapeutic potential may extend outside of ß-amyloid reliance. The described in vitro and cell based-in vitro coupled enzymatic assay systems proved suitable platforms to screen a preliminary library of new analogs.

Small Amphiphilic Peptides: Activity Against a Broad Range of Drug-Resistant Bacteria and Structural Insight into Membranolytic Properties.

We report the synthesis and antibacterial activities of a series of amphiphilic membrane-active peptides composed, in part, of various nongenetically coded hydrophobic amino acids. The lead cyclic peptides, 8C and 9C, showed broad-spectrum activity against drug-resistant Gram-positive (minimum inhibitory concentration (MIC) = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria. The cytotoxicity study showed the predominant lethal action of the peptides against bacteria as compared with mammalian cells. A plasma stability study revealed approximately 2-fold higher stability of lead cyclic peptides as compared to their linear counterparts after 24 h of incubation. A calcein dye leakage experiment revealed the membranolytic effect of the cyclic peptides. Nuclear magnetic resonance spectroscopy and molecular dynamics simulation studies of the interaction of the peptides with the phospholipid bilayer provided a solid structural basis to explain the membranolytic action of the peptides with atomistic details. These results highlight the potential of newly designed amphiphilic peptides as the next generation of peptide-based antibiotics.

Cyclic Peptide-Gadolinium Nanocomplexes as siRNA Delivery Tools.

We have recently reported that a cyclic peptide containing five tryptophan, five arginine, and one cysteine amino acids [(WR)5C], was able to produce peptide-capped gadolinium nanoparticles, [(WR)5C]-GdNPs, in the range of 240 to 260 nm upon mixing with an aqueous solution of GdCl3. Herein, we report [(WR)5C]-GdNPs as an efficient siRNA delivery system. The peptide-based gadolinium nanoparticles (50 µM) did not exhibit significant cytotoxicity (~93% cell viability at 50 µM) in human leukemia T lymphoblast cells (CCRF-CEM) and triple-negative breast cancer cells (MDA-MB-231) after 48 h. Fluorescence-activated cell sorting (FACS) analysis indicated that the cellular uptakes of Alexa-488-labeled siRNA were found to be enhanced by more than 10 folds in the presence of [(WR)5C]-GdNPs compared with siRNA alone in CCRF-CEM and MDA-MB-231 cells after 6 h of incubation at 37 °C. The gene silencing efficacy of the nanoparticles was determined via the western blot technique using an over-expressed gene, STAT-3 protein, in MDA-MB-231 cells. The results showed ~62% reduction of STAT-3 was observed in MDA-MB-231 with [(WR)5C]-GdNPs at N/P 40. The integrity of the cellular membrane of CCRF-CEM cells was found to be intact when incubated with [(WR)5C]-Gd nanoparticles (50 µM) for 2 h. Confocal microscopy reveals higher internalization of siRNA in MDA-MB-231 cells using [(WR)5C]-GdNPs at N/P 40. These results provided insight about the use of the [(WR)5C]-GdNPs complex as a potent intracellular siRNA transporter that could be a nontoxic choice to be used as a transfection agent for nucleic-acid-based therapeutics.

Synthesis, characterization, and cytotoxicity evaluation of dextran-myristoyl-ECGKRK peptide conjugate.

CGKRK is a well-known tumor homing peptide with significant specificity for many types of cancer tissues. Herein, we describe the synthesis of a novel drug delivery system based on dextran decorated with myristoyl-ECGKRK peptide. The myristoylated peptide was synthesized and conjugated to dextran via an ester bond followed by purification. FT-IR and NMR confirmed the success of the conjugation reaction, while the surface morphology examination revealed that the conjugate has a characteristic porous network-like structure. Dynamic-light scattering measurements indicated the ability of the conjugate to self-assemble into nanoparticles with an average size of 248 ± 6.33 nm, and zeta potential of 10.7 mV. The cytotoxicity profiles for the peptide, dextran (Dex0), and dextran-peptide conjugate (Dex1) were evaluated against triple-negative breast cancer cells (MDA-MB-231), breast cancer cells (MCF-7), and human embryonic normal kidney cells (HEK-293). The results revealed that myristoyl-ECGKRK was noncytotoxic on the two different breast cancer cell lines up to 50 µM, but the cell viability was minimally reduced to 85% at 50 µm in HEK-293 cells. Similarly, Dex0 showed a neglected cytotoxicity profile at all tested concentrations. The Dex1 was not toxic to the cells up to a concentration of 8.3 mg/mL.

Cyclic Dipeptides: The Biological and Structural Landscape with Special Focus on the Anti-Cancer Proline-Based Scaffold.

Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of peptides widespread in nature, are unsurpassed in their structural and bio-functional diversity. DKPs, especially those containing proline, due to their unique features such as, inter alia, extra-rigid conformation, high resistance to enzyme degradation, increased cell permeability, and expandable ability to bind a diverse of targets with better affinity, have emerged in the last years as biologically pre-validated platforms for the drug discovery. Recent advances have revealed their enormous potential in the development of next-generation theranostics, smart delivery systems, and biomaterials. Here, we present an updated review on the biological and structural profile of these appealing biomolecules, with a particular emphasis on those with anticancer properties, since cancers are the main cause of death all over the world. Additionally, we provide a consideration on supramolecular structuring and synthons, based on the proline-based DKP privileged scaffold, for inspiration in the design of compound libraries in search of ideal ligands, innovative self-assembled nanomaterials, and bio-functional architectures.

Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positively Charged and Hydrophobic Residues as Molecular Transporters.

The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW)5]K(RW)X (X = 1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 µM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), Stavudine (F'-d4T)), where F' is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 µM) was the most efficient transporter that improved the cellular uptake of F'-GpYEEI (2 µM) by 18- and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F'-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescence-labeled peptide (F'-[(RW)5K](RW)5) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.

Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity.

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R5K]W7A and linear peptide R5KW7A were conjugated with Dox through a glutarate linker to afford [R5K]W7A-Dox and R5KW7A-Dox conjugates to generate Dox derivatives. Alternatively, [R5K]W7C was conjugated with Dox via a disulfide linker to generate [R5K]W7C-S-S-Dox conjugate, where S-S is a disulfide bond. Comparative antiproliferative assays between conjugates [R5K]W7A-Dox, [R5K]W7C-S-S-Dox, linear R5KW7A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R5K]W7A-Dox conjugate was 2-fold more efficient than R5KW7A-Dox, and [R5K]W7C-S-S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R5K]W7A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 µM after 72 h of incubation, which was comparable to Dox (5 µM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R5K]W7A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5-7%) and heart cells (H9C2, <9%) at concentrations of 5-10 µM (compared to free Dox at 5 µM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [R5K]W7A-Dox (5 µM) in H9C2 cells. In comparison, Dox (5 µM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [R5K]W7A-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC50 of 2.3 and 4.3 µM, respectively) compared to free Dox (LC50 of 36-41 µM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R5K]W7A-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [R5K]W7A-Dox.

Synthesis and antiviral activity of fatty acyl conjugates of remdesivir against severe acute respiratory syndrome coronavirus 2 and Ebola virus.

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 µM; IC50(Calu3) = 0.24 µM), 3'-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 µM; IC50(Calu3) = 0.18 µM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 µM; IC50(Calu3) = 0.25 µM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 µM; IC50(Calu3) = 0.06 µM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.

Cyclic Peptides as Protein Kinase Inhibitors: Structure-Activity Relationship and Molecular Modeling.

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR]5, a peptide containing alternative arginine (R) and tryptophan (W) residues as a non-competitive c-Src tyrosine kinase inhibitor. A number of larger cyclic peptides containing alternative hydrophobic and positively charged residues [WR]x (x = 6-9) and hybrid cyclic-linear peptides, [R6K]W6 and [R5K]W7, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR]9 was found to be the most potent tyrosine kinase inhibitor. [WR]9 showed higher inhibitory activity (IC50 = 0.21 µM) than [WR]5, [WR]6, [WR]7, and [WR]8 with IC50 values of 0.81, 0.57, 0.35, and 0.33 µM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-33P]ATP. Consistent with the result above, [WR]9 inhibited other protein kinases such as Abl kinase activity with an IC50 value of 0.35 µM, showing 2.2-fold higher inhibition than [WR]5 (IC50 = 0.79 µM). [WR]9 also inhibited PKCa kinase activity with an IC50 value of 2.86 µM, approximately threefold higher inhibition than [WR]5 (IC50 = 8.52 µM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR]9 exhibited IC50 values of <0.25 µM against Akt1, Alk, and Btk. These data suggest that [WR]9 is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [R6K]W6 and [R5K]W7 against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR](5-9). The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR]5 or [WR]9 affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR]9.