Doxorubicin-Loaded Polyelectrolyte Multilayer Capsules Modified with Antitumor DR5-Specific TRAIL Variant for Targeted Drug Delivery to Tumor Cells.

Recently, biodegradable polyelectrolyte multilayer capsules (PMC) have been proposed for anticancer drug delivery. In many cases, microencapsulation allows to concentrate the substance locally and prolong its flow to the cells. To reduce systemic toxicity when delivering highly toxic drugs, such as doxorubicin (DOX), the development of a combined delivery system is of paramount importance. Many efforts have been made to exploit the DR5-dependent apoptosis induction for cancer treatment. However, despite having a high antitumor efficacy of the targeted tumor-specific DR5-B ligand, a DR5-specific TRAIL variant, its fast elimination from a body limits its potential use in a clinic. A combination of an antitumor effect of the DR5-B protein with DOX loaded in the capsules could allow to design a novel targeted drug delivery system. The aim of the study was to fabricate PMC loaded with a subtoxic concentration of DOX and functionalized with the DR5-B ligand and to evaluate a combined antitumor effect of this targeted drug delivery system in vitro. In this study, the effects of PMC surface modification with the DR5-B ligand on cell uptake both in 2D (monolayer culture) and 3D (tumor spheroids) were studied by confocal microscopy, flow cytometry and fluorimetry. Cytotoxicity of the capsules was evaluated using an MTT test. The capsules loaded with DOX and modified with DR5-B demonstrated synergistically enhanced cytotoxicity in both in vitro models. Thus, the use of the DR5-B-modified capsules loaded with DOX at a subtoxic concentration could provide both targeted drug delivery and a synergistic antitumor effect.

Fabrication and evaluation of nanocontainers for lipophilic anticancer drug delivery in 3D in vitro model.

Presently, most of anticancer drugs are high toxic for normal cells and, and as a result, they have severe side effects. Moreover, most of the formulations are lipophilic and have poor selectivity. To overcome these limitations, various drug delivery systems could be proposed. The aim of the current study was to fabricate novel polysaccharide nanocontainers (NC) by one-step ultrasonication technique and to evaluate their accumulation efficacy and cytotoxicity in 2D (monolayer culture) and 3D (tumor spheroids) in vitro models. NC with mean sizes in a range of 340-420 nm with the core-shell structure are synthetized and characterized. The NC shell is composed from diethylaminoethyl dextran/xanthan gum polyelectrolyte complex, while the hydrophobic core was loaded with the lipophilic anticancer drug thymoquinone. To enhance NC accumulation in human breast adenocarcinoma MCF-7 cells, the NC surface was modified with poly-L-lysine (PLL) or polyethylene glycol. Cell uptake of the NC loaded with Nile Red into the tumor cells was investigated by laser scanning confocal microscopy, fluorescent flow cytometry and fluorimetry. Modification of the NC with PLL allowed to obtain the optimal drug delivery system with maximal cytotoxicity, which was tested by MTT-test. The developed NC are promising for lipophilic anticancer drug delivery.

Novel bexarotene derivatives: Synthesis and cytotoxicity evaluation for glioma cells in 2D and 3D in vitro models.

Glioblastoma (GBM) is an aggressive and lethal form of brain cancer with a high invasion capacity and a lack of effective chemotherapeutics. Retinoid bexarotene (BXR) inhibits the neurospheroidal colony formation and migration of primary glioblastoma cells but has side effects. To enhance the BXR glioblastoma selectivity and cytotoxicity, we chemically modified it at the carboxyl group with either nitroethanolamine (NEA) bearing a NO-donating group (a well-known bioactivity enhancer; BXR-NEA) or with a dopamine (DA) moiety (to represent the highly toxic for various tumor cells N-acyldopamine family; BXR-DA). These two novel compounds were tested in the 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Both BXR-DA and BXR-NEA were found to be more toxic for rat C6 and human U-87MG glioma cells than the initial BXR. After 24 h incubation of the cells (monolayer culture) with the drugs, the IC50 values were in the range of 28-42, and 122-152 µM for BXR derivatives and BXR, respectively. The cell death occurred via apoptosis according to the annexin staining and caspase activation. The tumor spheroids demonstrated higher resistance to the treatment compared to that one of the monolayer cultures. BXR-DA and BXR-NEA were more specific against tumor cells than the parental drug, in particular the selectivity index was 1.8-2.7 vs. 1.3-1.5, respectively. Moreover, they inhibited cell migration more effectively than parental BXR according to a scratch assay. Cell spreading from the tumor spheroids was also inhibited. Thus, the obtained BXR derivatives could be promising for glioblastoma treatment.

A Triple Combination of Metformin, Acetylsalicylic Acid, and Oseltamivir Phosphate Impacts Tumour Spheroid Viability and Upends Chemoresistance in Triple-Negative Breast Cancer.

INTRODUCTION: Targeted multimodal approaches need to be strategically developed to control tumour growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes that arise. The tumour stage and cellular subtypes often dictate the appropriate clinical treatment regimen. Also, the development of chemoresistance is a common clinical challenge with breast cancer. Higher doses and additional drug agents can produce additional adverse effects leading to a more aggressive malignancy. Acetylsalicylic acid (ASA), metformin (Met), and oseltamivir phosphate (OP) were investigated for their efficacy to sensitize MDA-MB-231 triple-negative breast cancer and its tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR) together in combination with Tmx treatment. METHODS: Microscopic imaging, the formation of 3D multicellular tumour spheroids, immunocytochemistry, flow cytometry, Annexin V Assay, Caspase 3/7 Apoptosis Assay, tube formation assay and analysis, and WST-1 cell viability assay evaluated the formation of MCTS, morphologic changes, cell viability, apoptosis activity and the expression levels of ALDH1A1, CD44 and CD24 on the cell surface, MDA-MB231 triple-negative breast cancer, tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR). RESULTS: The results using a triple combination of ASA, Met and OP on MDA-MB-231 and MDA-MB-231-TmxR cells and their matrix-free 3D multicellular tumour spheroids (MCTS) formed by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method demonstrate a consistent and significant decrease in cell and tumour spheroid viability and volume with increased apoptotic activity, and increased sensitivity to Tmx therapy. Tmx treatment of MDA-MB-231 cells in combination with ASA, Met and OP markedly reduced the CD44/CD24 ratio by 6.5-fold compared to the untreated control group. Tmx treatment of MDA-MB-231-TmxR cells in combination with ASA, Met and OP markedly reduced the ALDH1A1 by 134-fold compared to the same treatment for the parental cell line. Also, the triple combination treatment of ASA, Met, and OP inhibited vasculogenic endothelial cell tube formation and induced endothelial cell apoptosis. CONCLUSION: For the first time, the findings demonstrate that repurposing ASA, Met, and OP provides a novel and promising targeted multimodal approach in the treatment of triple-negative breast cancer and its chemoresistant variant.

Impact of Fucosylation on Self-Assembly of Prostate and Breast Tumor Spheroids by Using Cyclo-RGDfK(TPP) Peptide and Image Object Detection.

INTRODUCTION: Core fucosylation of N-glycans on the integrin ß1 subunit is essential for the functional activity of the integrin. The binding of α5ß1 integrin with the tripeptide Arg-Gly-Asp (RGD) motif within the extracellular matrix protein fibronectin may be influenced by the α-1,6-fucose core or α-1,2-fucose and α-1,3/4-fucose peripheral N-glycan profiles. Here, we investigated whether fucosylation impacts the formation of matrix-free 3D multicellular tumor spheroids (MCTS) from human triple negative breast MDA-MB231 cell line, prostate PC3 and DU145 cell lines and DU145 gemcitabine resistant (GemR) variant by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method. METHODS: Microscopic imaging, lectin histochemistry, flow cytometry, WST-1 cell viability assay and You Only Look Once version 2 (YOLOv2) training object detection using cyclic learning rates were used to evaluate the formation of MCTS, morphologic changes, and the expression levels of α-1,6-fucose and α-1,2-fucose linkages on the cell surface. RESULTS: DU145 prostate cancer cells expressed higher α-1,6-fucose than α-1,2-fucose linkages on their cell surface, as determined by lectin cytochemistry and flow cytometry. Blockage of the α-1,6- and α-1,2-fucose linkages with Aspergillus oryzae lectin (AOL) and Ulex Europaeus agglutinin I (UEA I) one hour before the addition of cyclic-RGDfK(TPP) peptide to the monolayer of the cancer cells resulted in a statistically significant dose-dependent reduction in spheroid volumes using threshold diameters of 40 and 60 µm. Application of a 40 µm threshold diameter measurements of spheroids resulted in fewer false-positive ones compared to the 60 µm diameter threshold previously used in our studies. A state-of-the-art, image object detection system YOLOv2 was used to automate the analysis of spheroid measurements and volumes. The results showed that YOLOv2 corroborated manual spheroid detection and volume measurements with high precision and accuracy. CONCLUSION: For the first time, the findings demonstrate that α-1,6- and α-1,2-fucose linkages of N-glycans on the cell surface receptors facilitate cyclo-RGDfK(TPP)-mediated self-assembly of cancer cells to form 3D multicellular tumor spheroids.

Metformin increases antitumor activity of MEK inhibitor binimetinib in 2D and 3D models of human metastatic melanoma cells.

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.

Functionalized Folic Acid-Conjugated Amphiphilic Alternating Copolymer Actively Targets 3D Multicellular Tumour Spheroids and Delivers the Hydrophobic Drug to the Inner Core.

Engineering of a "smart" drug delivery system to specifically target tumour cells has been at the forefront of cancer research, having been engineered for safer, more efficient and effective use of chemotherapy for the treatment of cancer. However, selective targeting and choosing the right cancer surface biomarker are critical for a targeted treatment to work. Currently, the available delivery systems use a two-dimensional monolayer of cancer cells to test the efficacy of the drug delivery system, but designing a "smart" drug delivery system to be specific for a tumour in vivo and to penetrate the inner core remains a major design challenge. These challenges can be overcome by using a study model that integrates the three-dimensional aspect of a tumour in a culture system. Here, we tested the efficacy of a functionalized folic acid-conjugated amphiphilic alternating copolymer poly(styrene-alt-maleic anhydride) (FA-DABA-SMA) via a biodegradable linker 2,4-diaminobutyric acid (DABA) to specifically target and penetrate the inner core of three-dimensional avascular human pancreatic and breast tumour spheroids in culture. The copolymer was quantitatively analyzed for its hydrophobic drug encapsulation efficiency using three different chemical drug structures with different molecular weights. Their release profiles and tumour targeting properties at various concentrations and pH environments were also characterized. Using the anticancer drug curcumin and two standard clinical chemotherapeutic hydrophobic drugs, paclitaxel and 5-fluorouracil, we tested the ability of FA-DABA-SMA nanoparticles to encapsulate the differently sized drugs and deliver them to kill monolayer pancreatic cancer cells using the WST-1 cell proliferation assay. The findings of this study revealed that the functionalized folic acid-conjugated amphiphilic alternating copolymer shows unique properties as an active "smart" tumor-targeting drug delivery system with the ability to internalize hydrophobic drugs and release the chemotherapeutics for effective killing of cancer cells. The novelty of the study is the first to demonstrate a functionalized "smart" drug delivery system encapsulated with a hydrophobic drug effectively targeting and penetrating the inner core of pancreatic and breast cancer spheroids and reducing their volumes in a dose- and time-dependent manner.

Sialylation facilitates self-assembly of 3D multicellular prostaspheres by using cyclo-RGDfK(TPP) peptide.

BACKGROUND: Prostaspheres-based three dimensional (3D) culture models have provided insight into prostate cancer (PCa) biology, highlighting the importance of cell-cell interactions and the extracellular matrix (EMC) in the tumor microenvironment. Although these 3D classical spheroid platforms provide a significant advance over 2D models mimicking in vivo tumors, the limitations involve no control of assembly and structure with only limited spatial or glandular organization. Here, matrix-free prostaspheres from human metastatic prostate carcinoma PC3 and DU145 cell lines and their respective gemcitabine resistant (GemR) variants were generated by using cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)). MATERIALS AND METHODS: Microscopic imaging, immunocytochemistry (ICC), flow cytometry, sialidase, and WST-1 cell viability assays were used to evaluate the formation of multicellular tumor spheroid (MCTS), cell survival, morphologic changes, and expression levels of α2,6 and α2,3 sialic acid (SA) and E- and N-cadherin in DU145, PC3, and their GemR variants. RESULTS: By using the cyclo-RGDfK(TPP) peptide platform in a dose- and time-dependent manner, both DU145 and DU145GemR cells formed small MCTS. In contrast, PC3 and PC3GemR cells formed irregular multicellular aggregates at all concentrations of cyclo-RGDfK(TPP) peptide, even after 6 days of incubation. ICC and flow cytometry results revealed that DU145 cells expressed higher amounts of E-cadherin but lower N-cadherin compared with PC3 cells. By using Maackia amurensis (α2,3-SA-specific MAL-II) and Sambucus nigra (α2,6-SA specific SNA) lectin-based cytochemistry staining and flow cytometry, it was found that DU145 and DU145GemR cells expressed 5 times more α2,6-SA than α2,3-SA on the cell surface. PC3 cells expressed 4 times more α2,3-SA than α2,6-SA, and the PC3GemR cells showed 1.4 times higher α2,6-SA than α2,3-SA. MCTS volume was dose-dependently reduced following pretreatment with α2,6-SA-specific neuraminidase (Vibrio cholerae). Oseltamivir phosphate enhanced cell aggregation and compaction of 3D MCTS formed with PC3 cells. CONCLUSION: The relative levels of specific sialoglycan structures on the cell surface correlate with the ability of PCa cells to form avascular multicellular prostaspheres.

Novel Doxorubicin Derivatives: Synthesis and Cytotoxicity Study in 2D and 3D in Vitro Models.

Purpose: Multidrug resistance (MDR) of tumors to chemotherapeutics often leads to failure of cancer treatment. The aim of the study was to prepare novel MDR-overcoming chemotherapeutics based on doxorubicin (DOX) derivatives and to evaluate their efficacy in 2D and 3D in vitro models. Methods: To overcome MDR, we synthesized five DOX derivatives, and then obtained non-covalent complexes with human serum albumin (HSA). Drug efficacy was evaluated for two tumor cell lines, namely human breast adenocarcinoma MCF-7 cells and DOX resistant MCF-7/ADR cells. Additionally, MCF-7 cells were entrapped in alginate-oligochitosan microcapsules, and generated tumor spheroids were used as a 3D in vitro model to study cytotoxicity of the DOX derivatives. Results: Due to 3D structure, the tumor spheroids were more resistant to chemotherapy compared to monolayer culture. DOX covalently attached to palmitic acid through hydrazone linkage (DOX-N2H-Palm conjugate) was found to be the most promising derivative. Its accumulation levels within MCF-7/ADR cells was 4- and 10-fold higher than those of native DOX when the conjugate was added to cultivation medium without serum and to medium supplemented with 10% fetal bovine serum, respectively. Non-covalent complex of the conjugate with HSA was found to reduce the IC50 value from 32.9 µM (for free DOX-N2H-Palm) to 16.8 µM (for HSA-DOX-N2H-Palm) after 72 h incubation with MCF-7/ADR cells. Conclusion: Palm-N2H-DOX conjugate was found to be the most promising DOX derivative in this research. The formation of non-covalent complex of Palm-N2H-DOX conjugate with HSA allowed improving its anti-proliferative activity against both MCF-7 and MCF-7/ADR cells.

Oxime ligation in acetic acid: efficient synthesis of aminooxy-peptide conjugates.

Oxime ligation is a powerful tool in various bioconjugation strategies. Nevertheless, high reaction rates and quantitative yields are typically reported for aldehyde-derived compounds. In contrary, keto groups react much slower, with quantitative yields achieved at 5 h for low-molecular weight compounds and more than 15 h for polymers or dendrimers. In this communication, we report that oxime ligation proceeds rapidly with quantitative (>95%) conversion within 1.5-2 h in pure acetic acid. The practical utility of suggested technique is illustrated by the synthesis of peptide-steroid and peptide-polymer conjugates of model aminooxy-peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

3D in vitro co-culture models based on normal cells and tumor spheroids formed by cyclic RGD-peptide induced cell self-assembly.

OBJECTIVES: To design novel 3D in vitro co-culture models based on the RGD-peptide-induced cell self-assembly technique. RESULTS: Multicellular spheroids from M-3 murine melanoma cells and L-929 murine fibroblasts were obtained directly from monolayer culture by addition of culture medium containing cyclic RGD-peptide. To reach reproducible architecture of co-culture spheroids, two novel 3D in vitro models with well pronounced core-shell structure from tumor spheroids and single mouse fibroblasts were developed based on this approach. The first was a combination of a RGD-peptide platform with the liquid overlay technique with further co-cultivation for 1-2 days. The second allowed co-culture spheroids to generate within polyelectrolyte microcapsules by cultivation for 2 weeks. M-3 cells (a core) and L-929 fibroblasts (a shell) were easily distinguished by confocal microscopy due to cell staining with DiO and DiI dyes, respectively. CONCLUSIONS: The 3D co-culture spheroids are proposed as a tool in tumor biology to study cell-cell interactions as well as for testing novel anticancer drugs and drug delivery vehicles.

Sialylation transmogrifies human breast and pancreatic cancer cells into 3D multicellular tumor spheroids using cyclic RGD-peptide induced self-assembly.

Multicellular tumor spheroids (MTS) have been at the forefront of cancer research, designed to mimic tumor-like developmental patterns in vitro. Tumor growth in vivo is highly influenced by aberrant cell surface-specific sialoglycan structures on glycoproteins. Aberrant sialoglycan patterns that facilitate MTS formation are not well defined. Matrix-free spheroids from breast MCF-7 and pancreatic PANC1 cancer cell lines and their respective tamoxifen (TMX) and gemcitabine (Gem) resistant variants were generated using the RGD platform of cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK (TPP)). MCF-7 and MCF-7 TMX cells formed tight spheroids both in the classical agarose-and RGD-based platforms while all PANC1 cells formed loose aggregates. Using lectin histochemistry staining, sialidase assay, neuraminidase (Vibrio cholerae) and oseltamivir phosphate (OP) neuraminidase inhibitor treatments, MCF-7 and PANC1 cells and their drug-resistant variants expressed different sialic acid (SA) content on their cell surfaces. α-2,3- and α-2,6-sialic acid surface residues facilitated spheroid formation under cyclo-RGDfK(TPP)-induced self-assembly. Pretreatment with α-2,3- SA specific Maackia amurensis (MAL-II) lectin, α-2,6-SA specific Sambucus nigra (SNA) lectin, and exogenous α-2,6-SA specific neuraminidase (Vibrio cholerae) dose-dependently reduced spheroid volume. OP enhanced cell aggregation and compaction forming spheroids. PANC1 and MDA-MB231 xenograft tumors from untreated and OP-treated RAGxCγ double mutant mice expressed significantly higher levels of α-2,3- SA over α-2,6-SA. MCF-7 spheroids also expressed a high α-2,3-SA to α-2,6-SA ratio. These results suggest that the relative levels of specific sialoglycan structures on the cell surface correlate with the ability of cancer cells to form avascular multicellular tumor spheroids and in vivo xenograft tumors.

Formation of multicellular tumor spheroids induced by cyclic RGD-peptides and use for anticancer drug testing in vitro.

Development of novel anticancer formulations is a priority challenge in biomedicine. However, in vitro models based on monolayer cultures (2D) which are currently used for cytotoxicity tests leave much to be desired. More and more attention is focusing on 3D in vitro systems which can better mimic solid tumors. The aim of the study was to develop a novel one-step highly reproducible technique for multicellular tumor spheroid (MTS) formation using synthetic cyclic RGD-peptides, and to demonstrate availability of the spheroids as 3D in vitro model for antitumor drug testing. Cell self-assembly effect induced by addition of both linear and cyclic RGD-peptides directly to monolayer cultures was studied for 12 cell lines of various origins, including tumor cells (e.i. U-87 MG, MCF-7, M-3, HCT-116) and normal cells, in particular L-929, BNL.CL2, HepG2. Cyclo-RGDfK and its modification with triphenylphosphonium cation (TPP), namely cyclo-RGDfK(TPP) in a range of 10-100µM were found to induce spheroid formation. The obtained spheroids were unimodal with mean sizes in a range of 60-120µm depending on cell line and serum content in culture medium. The spheroids were used as 3D in vitro model, in order to evaluate cytotoxicity effects of antitumor drugs (doxorubicin, curcumin, temozolomide). The developed technique could be proposed as a promising tool for in vitro test of novel antitumor drugs.

Ultrasonically Assisted Polysaccharide Microcontainers for Delivery of Lipophilic Antitumor Drugs: Preparation and in Vitro Evaluation.

High toxicity, poor selectivity, and severe side effects are major drawbacks of anticancer drugs. Various drug delivery systems could be proposed to overcome these limitations. The aim of this study was to fabricate polysaccharide microcontainers (MCs) loaded with thymoquinone (TQ) by a one-step ultrasonication technique and to study their cellular uptake and cytotoxicity in vitro. Two MC fractions with a mean size of 500 nm (MC-0.5) and 2 µM (MC-2) were prepared and characterized. Uptake of the MCs by mouse melanoma M-3 cells was evaluated in both 2D (monolayer culture) and 3D (multicellular tumor spheroids) models by confocal microscopy, flow cytometry, and fluorimetry. The higher cytotoxicity of the TQ-MC-0.5 sample than the TQ-MC-2 fraction was in good correlation with higher MC-0.5 accumulation in the cells. The MC-0.5 beads were more promising than the MC-2 particles because of a higher cellular uptake in both 2D and 3D models, an enhanced antitumor effect, and a lower nonspecific toxicity.

HSA-based phosphorescent probe for two-photon in vitro visualization.

Two-photon microscopy reveals several advantages over conventional one since it provides higher spatial resolution as well as deeper penetration into the sample under study. The development of suitable two-photon probes is one of the most challenging tasks in this area. Here we present phosphorescent non-covalent adduct of human serum albumin and Au-Ag alkynyl-diphosphine complex, [Au14Ag4(C2Ph)12(PPh2C6H4PPh2)6][PF6]4, which exhibits high cross section of two-photon-induced luminescence (δTPE) within large near-infrared excitation wavelength region (700-800 nm) with maximum δTPE about 38 GM at 740 nm. This feature makes it a promising probe for multiphoton bioimaging as demonstrated by successful visualization of glioma C6 cells and various tissues by two-photon confocal microscopy both in planar and z-stacking modes. Additionally, the broad excitation region enables optimization of the signal-to-background auto-fluorescence ratio via variation of excitation wavelength.

Creatinyl amino acids: new hybrid compounds with neuroprotective activity.

Prolonged oral creatine administration resulted in remarkable neuroprotection in experimental models of brain stroke. However, because of its polar nature creatine has poor ability to penetrate the blood-brain barrier (BBB) without specific creatine transporter (CRT). Thus, synthesis of hydrophobic derivatives capable of crossing the BBB by alternative pathway is of great importance for the treatment of acute and chronic neurological diseases including stroke, traumatic brain injury and hereditary CRT deficiency. Here we describe synthesis of new hybrid compounds-creatinyl amino acids, their neuroprotective activity in vivo and stability to degradation in different media. The title compounds were synthesized by guanidinylation of corresponding sarcosyl peptides or direct creatine attachment using isobutyl chloroformate method. Addition of lipophilic counterion (p-toluenesulfonate) ensures efficient creatine dissolution in DMF with simultaneous protection of guanidino group towards intramolecular cyclization. It excludes the application of expensive guanidinylating reagents, permits to simplify synthetic procedure and adapt it to large-scale production. The biological activity of creatinyl amino acids was tested in vivo on ischemic stroke and NaNO(2) -induced hypoxia models. One of the most effective compounds-creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. These data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment.

Incorporation of N-amidino-pyroglutamic acid into peptides using intramolecular cyclization of alpha-guanidinoglutaric acid.

N-terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N-amidino-amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block-N-amidino-pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N-amidino-proline using RuO(4) did not produce positive results, N-amidino-Glp-Phe-OH was synthesized on Wang polymer by cyclization of alpha-guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N-amidino-Glp-Phe-OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N-amidino-Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach.

Protective effects of some creatine derivatives in brain tissue anoxia.

Some derivatives more lipophylic than creatine, thus theoretically being capable to better cross the blood-brain barrier, were studied for their protective effect in mouse hippocampal slices. We found that N-amidino-piperidine is harmful to brain tissue, and that phosphocreatine is ineffective. Creatine, creatine-Mg-complex (acetate) and phosphocreatine-Mg-complex (acetate) increased the latency to population spike disappearance during anoxia. Creatine and creatine-Mg-complex (acetate) also increased the latency of anoxic depolarization, while the delay induced by phosphocreatine-Mg-complex (acetate) was of borderline significance (P = 0.056). Phosphocreatine-Mg-complex (acetate) significantly reduced neuronal hyperexcitability during anoxia, an effect that no other compound (including creatine itself) showed. For all parameters except reduced hyperexcitability the effects statistically correlated with tissue levels of creatine or phosphocreatine. Summing up, exogenous phosphocreatine and N-amidino piperidine are not useful for brain protection, while chelates of both creatine and phosphocreatine do replicate some of the known protective effects of creatine. In addition, phosphocreatine-Mg-complex (acetate) also reduced neuronal hyperexcitability during anoxia.

Complexes of plasmid DNA with basic domain 47-57 of the HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways.

Arginine-rich peptides, penetratins, as part of a number of cellular and viral proteins, can penetrate across plasma membrane directly, without participation of endocytosis. We show that one of penetratins, the basic domain 47-57 of human immunodeficiency virus, type 1, transcription factor Tat (Tat peptide), is able to interact with plasmid DNA electrostatically. These interactions result in formation of polyelectrolytic complexes at various negative/positive charge ratios of plasmid DNA and Tat peptide. Plasmid DNA is capable of binding to Tat peptide up to 1.7-fold excess of the complex positive charge. The DNA-Tat complexes can be used for delivery of plasmid DNA into mammalian cells. Transfection efficacy of cultured cells by DNA-Tat complexes is stimulated by free Tat peptide, most likely because it protects DNA-Tat complexes from disruption by anionic proteoglycans of cellular surface. Our data strongly argue in favor of the endocytosis-dependent mechanism of DNA-Tat complex uptake by mammalian cells similarly to internalization of complexes of plasmid DNA with other polycationic carriers. Moreover, different cell lines use different endocytosis-mediated pathways for DNA-Tat complex internalization. Intravenous injections to mice of DNA-Tat complexes in comparison with injections of naked DNA showed an inhibitory effect of DNA-Tat complex positive charge on expression of transferred gene. A low level of foreign gene expression in the liver of mice injected intravenously with positively charged DNA-Tat complexes is accounted for by inactivation of DNA-Tat complexes in the bloodstream due to their interactions with serum albumin. These data should be taken into account in an attempt to develop versatile gene delivery systems based on penetratin application for human disease therapy.