Poly L-aspartic Acid Research Articles

Bulk Biopolyelectrolyte Complexes from Homopolypeptides: Solid "Salt Bridges".

Salt bridges, pairings between oppositely charged amino acids, are dispersed throughout proteins to assist folding and interactions. Biopolyelectrolyte complexes (BioPECs) were made between the homopolypeptides poly-l-arginine (PLR) and poly-l-lysine (PLK) with sodium triphosphate (STPP), as well as from polypeptide-only combinations. Viscoelastic measurements on these high salt bridge density materials showed many were solid, even glassy, in nature. Although the polypeptide-phosphate complexes had similar moduli at room temperature, the PLR-STPP complex displayed an unusual melting event above 70 °C not seen in PLK-STPP. This event was supported with differential scanning calorimetry. Infrared spectroscopy showed the PLK-STPP system contained β-sheets, while PLR-STPP did not. Stoichiometric, macroscopic BioPECs of PLR and PLK with poly-l-aspartic acid (PLD) and poly-l-glutamic acid (PLE) were made. PLR-PLD was found to undergo a melting event similar to that in PLR-STPP. ATR-FTIR studies showed that BioPECs made with PLD do not contain β-sheets, while those composed of PLE do. This work illustrates an expanded palette of unique properties from these biomaterials, such as strong viscoelastic differences between PECs containing PLE and PLD, even though they differ by only one carbon on the side chain.

Poly-L-aspartic acid based nonconventional luminescent biomacromolecules with efficient emission in dilute solutions for Al3+ detection

Nonconventional luminescent macromolecules exhibiting bright fluorescence or phosphorescence emission at high concentrations and solid-state have attracted significant attention due to their promising application in different fields. However, most reported nonconventional luminescent macromolecules show weak or no emission in dilute solutions, limiting their large-scale applications. Herein, nonconventional luminescent biomacromolecules with hydrophobic rigid chains, hydrophilic flexibility and inter- or intra-molecular hydrogen bonding interactions were proposed to achieve effective luminescence in dilute solutions. Poly-L-aspartic acid (PASA) with a fluorescence quantum yield of 4.6 % in a dilute solution (0.8 mg/mL) was synthesized to validate this design strategy. The fluorescence intensity of PASA solution increased with the increase of the concentration, demonstrating a clustering-triggered emission (CTE) effect. Furthermore, the fluorescence intensity significantly enhanced when adding Al3+ into PASA aqueous solution via the Al3+ recognition effect. The detection limits for Al3+ (1.71 × 10−6 mol/L) meet the World Health Organization (WHO) requirements for food detection. At last, PASA solid-state samples exhibit room temperature phosphorescence emission.

SCIDOT-28. TARGETED NANOPARTICLES FOR IMPROVED DRUG DELIVERY TO MEDULLOBLASTOMA

Abstract Platinum-based agents remain a key component of therapy for children with medulloblastoma, despite significant systemic side effects and only modest blood-brain barrier (BBB) penetration. Cisplatin has a cerebrospinal fluid-to-plasma ratio <5% and dose-limiting side effects of nephrotoxicity, ototoxicity, and myelosuppression. Improving delivery of cisplatin across the BBB and selectively accumulating in tumors could improve its therapeutic index. To this end, we are leveraging chemical engineering techniques to rationally design cisplatin nanoparticles (NPs) to cross the BBB and preferentially enter medulloblastoma tumor cells. Using the layer-by-layer (LbL) platform to ‘wrap’ polyelectrolytes around a NP core by iterative electrostatic adsorption, we screened six negatively charged polypeptide and polysaccharide outer layers in medulloblastoma cell lines. Poly-L-aspartic acid (PLD) layered NPs had significant accumulation in tumor cells after 24 hours incubation, with an uptake index of 18±4 over unlayered control NPs. Next, we generated propargyl-functionalized PLD and used click chemistry to covalently conjugate the BBB shuttle ligands glutathione, angiopep-2, and transferrin, which have been shown to mediate transcytosis across brain endothelial cells. PLD layered NPs functionalized with angiopep-2 and transferrin had enhanced uptake in medulloblastoma tumor cells and NPs functionalized with glutathione were non-inferior to PLD layered NPs. After incubation with endothelial cells in vitro, all three BBB shuttle ligands enhanced uptake of PLD layered NPs over unlayered and non-functionalized control NPs. We then incorporated cisplatin into the nanoparticle core of this platform. Cisplatin-loaded NPs with PLD layering and ligand functionalization were more effective than free cisplatin as measured by IC50 over 72 hours in culture, and led to faster apoptosis as assessed by flow cytometry with annexin V and propidium iodide staining. In summary, functionalized nanoparticles are a promising platform to modulate drug delivery to medulloblastoma. In vivo studies using an orthotopic xenograft model are underway to investigate biodistribution, efficacy, and toxicity.

A Solid Phase Vibrational Circular Dichroism Study of Polypeptide-Surfactant Interaction.

We studied the interaction of poly-l-lysine (PLL) and poly-l-arginine (PLAG) with sodium dodecyl sulfate (SDS) surfactant and the interaction of poly-l-glutamic acid (PLGA) and poly-l-aspartic acid (PLAA) with tetradecyltrimethylammonium bromide (TTAB) surfactant using vibrational circular dichroism (VCD) spectroscopy in the region of C-H stretching vibration and in the Amide I region both in solution and in mulls. A chirality transfer from polypeptides to achiral surfactants was observed in the C-H stretching region, where measurements in solution were impossible. This observation was enabled by a special sample treatment technique using lyophilization and the preparation of mulls. This technique demonstrated itself as an interesting and beneficial tool for VCD measurements. In addition, we observed that SDS changed the secondary structure of PLL to the β-sheet and of PLAG to the α-helix. TTAB disrupted the PLGA and PLAA structure. These results were obtained in the mull but were confirmed by the VCD spectra measured in solution and by electronic circular dichroism. The chirality transfer from the polypeptides to SDS was caused by polypeptides ordered into a specific conformation during the interaction, while in the TTBA system it was induced primarily by the chirality of the amino acid residues.

Hyaluronic acid-conjugated apoferritin nanocages for lung cancer targeted drug delivery.

In this paper, we proposed a naturally derived protein cage based pH-responsive delivery system for intracellular prodrug controlled release. The drug delivery system is based on apoferritin as delivery vehicles to encapsulate the anticancer drug daunomycin (DN) and alleviate the side effect. The hydrophobic drug DN was encapsulated into the interior of apoferritin by the hydrophobic channels of the cage with swelling at slight acidic pH and electrostatic adsorption. The negatively charged poly-l-aspartic acid (PLAA) was further introduced into the apoferritin to absorb the positively charged DN. The mixture of PLAA and DN easily flew into the apoferritin cage and was stably stored in the physiological fluids. PLAA protected the leakage of DN and encapsulated a sufficient amount of drug molecules in the cage. To specifically target the tumor cells, the surface of apoferritin was modified with hyaluronic acid (HA) which can easily bind to the HA-receptor CD44. Here, human embryonic lung MRC-5 cells and lung cancer A549 cells were used to observe the specific binding of HA and morphological changes in vitro and examine the antitumor activity. This unique protein based drug delivery platform using the apoferritin cage shows great potential in the therapeutic administration of the anti-cancer agents.

Apoferritin-based nanomedicine platform for drug delivery: equilibrium binding study of daunomycin with DNA

This paper describes the preparation and optimization of the analytical properties of the protein based drug delivery platform apoferritin. In biological systems, the protein cage ferritin is used to store iron and to keep it from building to toxic levels in cells. When the iron atoms are removed from ferritin, apoferritin is formed. In this study, daunomycin, an anthracycline antibiotic drug that has been used for specific types of cancer treatment such as acute myeloid leukemia and acute lymphocytic leukemia, was encapsulated within the protein cage for drug delivery. Daunomycin slows or stops the growth of cancer cells by binding with the cell's DNA. The model for daunomycin-DNA complex binding mechanism is intercalation, where daunomycin binds with approximately every 3 base pairs causing a local unwinding, but a negligible distortion of the helical conformation. The binding affinity for free DNA is higher than that of structured DNA in cells. Upon binding with DNA the fluorescence intensity of daunomycin decreases. We used apoferritin's ability to disassemble and reassemble under pH control to load the therapeutic compound daunomycin. The combination of a modifiable interior and exterior surface and the passable hydrophobic and hydrophilic channels through the cage allows the containment or attachment of both insoluble and soluble drugs for delivery. At experimental pH 5 conditions the interaction between the apoferritin interior cage and daunomycin is weak making it difficult to encapsulate the drug effectively within the protein cage. The incorporation of poly-L-aspartic acid (PLAA), a polypeptide and biodegradable material that does not increase the toxicity of the drug delivery system and is negatively charged at pH 5.0, into the drug delivery system resulted in a substantial improvement in the drug encapsulation. The binding properties of free daunomycin with DNA were compared to the newly synthesized apoferritin protein based drug delivery system. Encapsulation of the daunomycin within the apoferritin protein cage had little effect upon the intrinsic binding constant, Ki, or the exclusion parameter n as compared to the free daunomycin model. The study resulted in the design and optimization of a unique protein based drug delivery platform using the protein cage apoferritin for potential therapeutic administration of the anti-cancer agent daunomycin.

Poly-l-aspartic Acid Enhances and Prolongs Gentamicin-mediated Suppression of the CFTR-G542X Mutation in a Cystic Fibrosis Mouse Model

Aminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-L-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20-40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr-/- hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach.

Effects of polyelectrolytic peptides on the quality of mineral crystals grown in vitro

Charged amino acids such as arginine, lysine, glutamic acid, and aspartic acid are abundant in noncollagenous proteins that regulate mineralization. Synthetic peptide forms of these amino acids have been shown to affect crystal growth in precipitation of mineral crystals in solution. However, little is known about the effects of these peptides on the viability and phenotype of bone marrow stromal cells (BMSCs) or on the in vitro mineralization process. Bone marrow was harvested from neonatal rat femora and cultured under conditions to induce mineralized nodule formation. Mineralized bone nodules were grown while supplementing the cultures with one of five polyelectrolytes: polystyrene sulfonate (PSS), poly-L: -glutamic acid (PLG), poly-L: -lysine (PLL), poly-L: -aspartic acid (PLA), and sodium citrate (SC), as well as a nontreated control group. The viability and the rate of collagen synthesis under the effect of these agents were characterized by cell-counting and dye-binding assays, respectively. Raman microspectroscopy was conducted on mineralized bone nodules to determine the effect of the polyelectrolytes on the mineralization, type-B carbonation, and crystallinity of the mineral phase. Morphology of resulting mineral crystals was investigated using X-ray diffraction line-broadening analysis (XRD). PSS had toxic effects on cells whereas the remaining agents were biocompatible, as the cell viability was either greater (PLG) or not different from controls. The total collagen production by day 21 was 27% and 42% lower than controls for PLL and PSS, respectively. Culture wells stained positively for alkaline phosphatase in the presence of polyelectrolytes, indicating that osteogenic differentiation was not impacted negatively. Raman microspectroscopy revealed that the type-B carbonation of the crystal lattice increased when treated with PLG, PLL, or PSS. Crystallinity of PLL and PSS was smaller than that of control. The mineral/matrix ratios of nodules did not change with polyelectrolyte treatment, with the exception of the PSS-treated group, which was less mineralized. XRD analysis of bone nodules indicated that PLA-treated samples were significantly longer than controls along the 002 direction. Overall, the results suggest that the polypeptides consisting of charged amino acids are biocompatible and that they have the potential to affect crystal quality and morphology in vitro in the presence of cells. However, the mechanisms by which these effects come into play remain to be investigated.

Platelet Separation From Whole Blood in an Aqueous Two-Phase System With Water-Soluble Polymers

Platelet-rich plasma (PRP) stimulates tissue healing and centrifugation is the only method for PRP preparation. The purpose of the present study was to develop a method to separate platelets without centrifugation. We used 16 polymers of different chemical characteristics and mixed each polymer with fresh whole blood containing anticoagulant citrate-dextrose. Then, we observed blood cell separation. Focusing on the effective polymers and comparing our method with the conventional centrifugation method, we examined platelet recovery rate and P-selectin expression, which represents platelet activation. Poly-L-glutamic acid (PGA) and poly-L-aspartic acid (PAA) separated platelets efficiently; however, these polymers activated platelets. On the contrary, poly (2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB), which has phosphorylcholine residues mimicking a cellular membrane molecule, separated platelets moderately and the PMB-separated platelets were nearly inactive compared to the separation with the ordinary centrifugation and PGA. Conclusively, the present experiments demonstrated that without centrifugation platelets can be separated from whole blood with some water-soluble polymers, such as PGA, PAA, and PMB, and that PMB has an advantage not to activate platelets.

Effect of Poly L-Aspartic Acid on the Biomimetic Formation of Calcium Phosphate on Collagen Gel

Effect of Poly L-Aspartic Acid on the Biomimetic Formation of Calcium Phosphate on Collagen Gel

A cationic lipid emulsion/DNA complex as a physically stable and serum-resistant gene delivery system.

To develop a non-viral gene delivery system in the form of an oil-in-water (o/w) lipid emulsion. Cationic lipid emulsions were formulated with soybean oil, 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as a cationic emulsifier and other co-emulsifiers. The physical characteristics of the lipid emulsion and the emulsion/DNA complex were determined. The in vitro transfection efficiency of the emulsion/DNA complex was determined in the presence of up to 90% serum. The average droplet size and zeta potential of emulsions were ca. 180 nm and ca. +50 mV, respectively. Among the emulsions, a stable formulation was selected to form a complex with a plasmid DNA encoding chloramphenicol acetyltransferase. By increasing the ratio of emulsion to DNA. zeta-potential of the emulsion/DNA complex increased monotonously from negative to positive without any changes in the complex size. The complex was stable against DNase I digestion and an anionic poly-L-aspartic acid (PLAA). The complex delivered DNA into the cells successfully, and the transfection efficiency was not affected by complex formation time from 20 min to 2 h. More importantly, the cationic lipid emulsion facilitated the transfer of DNA in the presence of up to 90% serum. The cationic lipid emulsion/DNA complex has physical stability and serum resistant properties for gene transfer.

Poly- l-aspartic acid protects cultured human proximal tubule cells against aminoglycoside-induced electrophysiological alterations

Cultured human proximal tubule cell monolayers maintained on permeable supports were treated simultaneously with the aminoglycoside antibiotic, gentamicin, and poly- l-aspartic acid (PAA), an inhibitor of aminoglycoside nephrotoxicity. Following 4 days of exposure, cell monolayers were placed into Ussing chambers to allow monitoring of transepithelial electrical properties. For each of the three cell isolatation examined, aminoglycoside-induced alterations in electrogenic transport, reflected by changes in short-circuit current (Isc), as well as alterations in paracellular properties, indicated by changes in transepithelial electrical resistance ( R T), were diminished in the presence of PAA. Alterations resulting from selective basolateral exposure to gentamicin were unchanged in the case of apically applied PAA and attenuated only when PAA acid was added basolaterally. This is the first demonstration of PAA inhibition of aminoglycoside-induced cellular alterations involving human cells.

Purification and characterization of the high molecule weight microtubule associated proteins from neonatal rat brain.

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highest levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide beta-II (422-434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the beta-II tubulin fragment, but it showed interaction with the Affigel-conjugated beta-I (431-444) tubulin peptide. The different MAPs components were characterized by western blots using specific monoclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Microtubule-Associated Proteins, MAP 1A and MAP IB, Interact with F-Actin In Vitro

Microtubule-associated protein (MAP) 1 consisting of MAP 1A and 1B was purified from rat brain by the poly-L-aspartic acid (PLAA) method. We found that MAP 1 bound to F-actin in vitro up to a molar ratio of MAP 1 to actin monomers of 1:10. The apparent binding constant was about 2.7 x 10(7) M-1. In contrast to the binding of MAP 2 or tau to F-actin, the binding of MAP 1 to F-actin did not affect the low-shear viscosity of actin filaments. Binding experiments performed using fragments of MAP 1, obtained by chymotrypsin digestion, indicated that MAP 1 included binding domains to F-actin that were different from those in microtubules and also two light chains (31 and 29 kDa) that were cosedimented with F-actin as well as with microtubules.