Poly L-glutamate Research Articles

Nanoengineered Polymeric RNA Nanoparticles for Controlled Biodistribution and Efficient Targeted Cancer Therapy.

RNA nanotechnology, including rolling circle transcription (RCT), has gained increasing interest as a fascinating siRNA delivery nanoplatform for biostable and tumor-targetable RNA-based therapies. However, due to the lack of fine-tuning technologies for RNA nanostructures, the relationship between physicochemical properties and siRNA efficacy of polymeric siRNA nanoparticles (PRNs) with different sizes has not yet been fully elucidated. Herein, we scrutinized the effects of size/surface chemistry-tuned PRNs on the biological and physiological interactions with tumors. PRNs with adjusted size and surface properties were prepared using sequential engineering processes: RCT, condensation, and nanolayer deposition of functional biopolymers. Through the RCT process, nanoparticles of three sizes with a diameter of 50-200 nm were fabricated and terminated with three types of biopolymers: poly-l-lysine (PLL), poly-l-glutamate (PLG), and hyaluronic acid (HA) for different surface properties. Among the PRNs, HA-layered nanoparticles with a diameter of ∼200 nm exhibited the most effective systemic delivery, resulting in superior anticancer effects in an orthotopic breast tumor model due to the CD44 receptor targeting and optimized nanosized structure. Depending on the type of PRNs, the in vivo siRNA delivery with protein expression inhibition differed by up to approximately 20-fold. These findings indicate that the types of layered biopolymers and the PRNs size mediate efficient polymeric siRNA delivery to the targeted tumors, resulting in high RNAi-induced therapeutic efficacy. This RNA-nanotechnology-based size/surface editing can overcome the limitations of siRNA therapeutics and represents a potent built-in module method to design RNA therapeutics tailored for targeted cancer therapy.

Electrostatic adsorption of polyanions onto lipid nanoparticles controls uptake, trafficking, and transfection of RNA and DNA therapies

Rapid advances in nucleic acid therapies highlight the immense therapeutic potential of genetic therapeutics. Lipid nanoparticles (LNPs) are highly potent nonviral transfection agents that can encapsulate and deliver various nucleic acid therapeutics, including but not limited to messenger RNA (mRNA), silencing RNA (siRNA), and plasmid DNA (pDNA). However, a major challenge of targeted LNP-mediated systemic delivery is the nanoparticles' nonspecific uptake by the liver and the mononuclear phagocytic system, due partly to the adsorption of endogenous serum proteins onto LNP surfaces. Tunable LNP surface chemistries may enable efficacious delivery across a range of organs and cell types. Here, we describe a method to electrostatically adsorb bioactive polyelectrolytes onto LNPs to create layered LNPs (LLNPs). LNP cores varying in nucleic acid cargo and component lipids were stably layered with four biologically relevant polyanions: hyaluronate (HA), poly-L-aspartate (PLD), poly-L-glutamate (PLE), and polyacrylate (PAA). We further investigated the impact of the four surface polyanions on the transfection and uptake of mRNA- and pDNA-loaded LNPs in cell cultures. PLD- and PLE-LLNPs increased mRNA transfection twofold over unlayered LNPs in immune cells. HA-LLNPs increased pDNA transfection rates by more than twofold in epithelial and immune cells. In a healthy C57BL/6 murine model, PLE- and HA-LLNPs increased transfection by 1.8-fold to 2.5-fold over unlayered LNPs in the liver and spleen. These results suggest that LbL assembly is a generalizable, highly tunable platform to modify the targeting specificity, stability, and transfection efficacy of LNPs, as well as incorporate other charged targeting and therapeutic molecules into these systems.

Ultrasensitive pH Triggered Charge/Size Dual-Rebound Gene Delivery System.

A facile strategy is developed to construct an ultrasensitive pH triggered charge/size dual-rebound gene delivery system for efficient tumor treatment. The therapeutic gene is complexed by polyethylenimine (PEI) and poly-l-glutamate (PLG), further in situ tightened by aldehyde modified polyethylene glycol (PEG) via Schiff base reaction. The generated Schiff base bonds are stable in neutral pH but cleavable in tumor extracellular pH. This gene delivery system possesses following favorable properties: (1) the tunable gene delivery system is constructed by chemical bench-free "green" and fast process which is favored by clinician, (2) PEG cross-linking shields the surface positive charges and tightens the complex particles, leading to decreased cytotoxicity, improved stability, and prolonged circulation, (3) PEG shielding can be rapidly peeled off by acidic pH as soon as arriving tumors, (4) dual charge/size ultrasensitively rebounding to higher positive potential and bigger size enhances tumor cell uptake efficiency. A series of experiments both in vitro and in vivo are carried out to investigate this gene delivery system in detail. An antiangiogenesis therapeutic gene is carried for the treatment of CT26 tumors in mice, achieving superior antitumor efficacy which is well proved by sufficient biological evidence. The system has great potentials for cancer therapy in the future.

In vivo magnetic resonance imaging of tumor protease activity.

Increased expression of cathepsins has diagnostic as well as prognostic value in several types of cancer. Here, we demonstrate a novel magnetic resonance imaging (MRI) method, which uses poly-L-glutamate (PLG) as an MRI probe to map cathepsin expression in vivo, in a rat brain tumor model. This noninvasive, high-resolution and non-radioactive method exploits the differences in the CEST signals of PLG in the native form and cathepsin mediated cleaved form. The method was validated in phantoms with known physiological concentrations, in tumor cells and in an animal model of brain tumor along with immunohistochemical analysis. Potential applications in tumor diagnosis and evaluation of therapeutic response are outlined.

Eosinophil peroxidase induces expression of cholinergic genes via cell surface neural interactions

Eosinophils localize to and release their granule proteins in close association with nerves in patients with asthma and rhinitis. These conditions are associated with increased neural function. In this study the effect of the individual granule proteins on cholinergic neurotransmitter expression was investigated. Eosinophil peroxidase (EPO) upregulated choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) gene expression. Fluorescently labeled EPO was seen to bind to the IMR-32 cell surface. Both Poly-l-Glutamate (PLG) and Heparinase-1 reversed the up-regulatory effect of EPO on ChAT and VAChT expression and prevented EPO adhesion to the cell surface. Poly-l-arginine (PLA) had no effect on expression of either gene, suggesting that charge is necessary but insufficient to alter gene expression. EPO induced its effects via the activation of NF-κB. MEK inhibition led to reversal of all up-regulatory effects of EPO. These data indicate a preferential role of EPO signaling via a specific surface receptor that leads to neural plasticity.

In vivo toxicity and biodistribution of intraperitoneal and intravenous poly-l-lysine and poly-l-lysine/poly-l-glutamate in rats

The combination of two differently charged polypeptides, poly-L-lysine (PL) and poly-L-glutamate (PG), has shown excellent postsurgical antiadhesive properties. However, the high molecular, positively charged PL is toxic in high doses, proposed as lysis of red blood cells. This study aims to elucidate the in vivo toxicity and biodistribution of PL and complex bound PLPG comparing intravenous and intraperitoneal administration. Fifty-six Sprague-Dawley rats were used in a model with repeated blood samples within 30 min examining blood gases and blood smears. Similarly, FITC labelled PL were used to track bio distribution and clearance of PL, given as single dose and complex bound to PG after intravenous and intraperitoneal administration. Tissue for histology and immunohistochemistry was collected. Blood gases and blood smears as well as histology points to a toxic effect of high dose PL given intravenously but not after intraperitoneal administration. The toxic effect is exerted through endothelial disruption and subsequent bleeding in the lungs, provoking sanguineous lung edema. FITC-labelled PL experiments reveal a rapid clearance with differences between routes and complex binding. This study advocates a new theory of the toxic effects in vivo of high molecular PL. PLPG complex is safe to use as antiadhesive prevention based on this toxicity study given that PL is always intraperitoneally administered in combination with PG and that the dose is adequate.

Molecular insights into the mechanisms of cation-type specific stability and denaturation of poly-l-glutamate: a simulation study

Cation-induced conformational changes of peptide as a guide to developing insights into human diseases-related proteins have received a lot of attention. The interactions between poly-l-glutamate (PGA) and different cations, including Na+, K+ and Mg2+, respectively, are studied in solvent at a concentration of 1 M, and the behaviours of peptide with different cations are investigated. For Na+, an oscillatory stabilising process to α-helix PGA is found, in accordance with the uniform free-energy landscape, whereas for K+, an extended α-helix structure is formed by the terminal turns, suggesting a weaker attraction to charged head groups. For Mg2+, the bridged charged side chains are responsible for the maximum probability of helix state. These distinct structural changes can be attributed to the different interactions between charged head groups and cations. Both Na+ and K+ are mainly attracted around head groups by direct ion binding while Mg2+ is centrally trapped among adjacent charged head groups. In addition, a surprising shift of the backbone hydrogen bond, from intact state to intermediate state, is observed. This is opposite to the stabilising effect of Na+ around negatively charged head groups.

Prevention of Pleural Adhesions by Bioactive Polypeptides - A Pilot Study

Objective: Postoperative pleural adhesions lead to major problems in repeated thoracic surgery. To date, no antiadhesive product has been proven clinically effective. Previous studies of differently charged polypeptides, poly-L-lysine (PL) and poly-L-glutamate (PG) have shown promising results reducing postoperative abdominal adhesions in experimental settings. This pilot study examined the possible pleural adhesion prevention by using the PL+PG concept after pleural surgery and its possible effect on key parameters; plasmin activator inhibitor-1 (PAI-1) and tissue growth factor beta 1 (TGFb) in the fibrinolytic process.Methods: A total of 22 male rats were used in the study, one control group (n=10) and one experimental group (n=12). All animals underwent primary pleural surgery, the controls receiving saline in the pleural cavity and the experimental group the PL+PG solution administered by spray. The animals were evaluated on day 7. Macroscopic appearance of adhesions was evaluated by a scoring system. Histology slides of the adhesions and pleural biopsies for evaluation of PAI-1 and TGFb1 were taken on day 7.Results: A significant reduction of adhesions in the PL+PG group (p<0.05) was noted at day 7 both regarding the length and severity of adhesions. There were no significant differences in the concentration of PAI-1 and TGFb1 when comparing the two groups.Conclusions: PL+PG may be used to prevent pleural adhesions. The process of fibrinolysis, and fibrosis was though not affected after PLPG administration.

Synthesis of Poly L-Glutamate Initiated by Triethanolamine

With L-glutamic acid as raw materials, N-carboxy-L-glutamic acid-benzyl-anhydride (BLG-NCA) was synthesized by triethanolamine. With triethanolamine as the initiator in the anhydrous dichloromethane, ring-opening polymerization of BLG-NCA was initiated, poly-l-glutamic acid benzyl ester was obtained. The amount of initiators, molar ratio of monomer/initiators, reaction temperature and reaction time to the effect of poly-l-glutamic acid benzyl ester in the ring-opening polymerization of BLG-NCA were studied. At last, poly L-glutamate was got, after the debenzylation in HBr solution. Through infrared spectroscopy, Ubbelohde viscometer, 1H NMR spectra, polymerization product was characterized and analysized. Results showed that in dichloromethane, ring-opening polymerization of BLG-NCA could be initiated by triethanolamine.

Synthesis of Poly L-Glutamate Initiated by Triethanolamine

With L-glutamic acid as raw materials, N-carboxy-L-glutamic acid-benzyl-anhydride (BLG-NCA) was synthesized by triethanolamine. With triethanolamine as the initiator in the anhydrous dichloromethane, ring-opening polymerization of BLG-NCA was initiated, poly-l-glutamic acid benzyl ester was obtained. The amount of initiators, molar ratio of monomer/initiators, reaction temperature and reaction time to the effect of poly-l-glutamic acid benzyl ester in the ring-opening polymerization of BLG-NCA were studied. At last, poly L-glutamate was got, after the debenzylation in HBr solution. Through infrared spectroscopy, Ubbelohde viscometer, 1H NMR spectra, polymerization product was characterized and analysized. Results showed that in dichloromethane, ring-opening polymerization of BLG-NCA could be initiated by triethanolamine.

Prevention of Adhesions by PL/PG after Adhesiolysis

Background: Previous studies of differently charged polypeptides, Poly-L-lysine (PL) and Poly-L-Glutamate (PG) have shown promising results, reducing postoperative adhesions. This study aimed to investigate the possible anti adhesion effect of those combined polypeptides, after adhesiolys. The concentration of tPA, PAI-1 and active TGFb1 in biopsies from adhesions, unharmed peritoneum before and after adhesiolysis, was also investigated. Materials and methods: A total of 24 male rats were divided in three groups A (N=8), B (N=8) and C (N=8). All rats underwent primary adhesion creating surgery at day 0, and adhesiolysis at day 7. Adhesions were evaluated at day 7 and 14, where group B received PL/PG after surgery at day 0 and after adhesiolysis at day 7, and group C received PL/PG after adhesiolysis at day 7. Tissue plasminogen activator (tPA), Plasminogen activator inhibitor 1(PAI-1) and active transforming growth factor beta 1(TGF-β1) were collected from biopsies of adhesions and normal peritoneum at day 0, 7 and 14. Results: Significant reduction of adhesions p<0.05 was seen in group B at day 7 after primary surgery, and at day 14 after adhesiolysis. Significantly p<0.05 reduction of adhesions was seen at day 14 after adhesiolysis in group C. Some variations were seen in tPA, PAI-1 and active TGFb1. Conclusions: PL/PG may be used to prevent adhesion formation after adhesiolysis. The process of fibrinolysis and fibrosis was not affected, after PL/PG prophylaxis and adhesiolysis.

Effects of Polylysine and Polyglutamate on Inflammation and the Normal Process of Peritoneal Healing After Surgery

Introduction: Intraperitoneal adhesions are common after abdominal surgery and may lead to serious clinical complications. Previous studies have investigated the possible effects of the polypeptides poly-L-lysine (αPL) and poly-L-glutamate (PG) forming a polymer complex that prohibits local peritoneal adhesions after surgery. The aim of this study was to examine whether the normal process of peritoneal healing was affected by PL/PG polymer matrix. Material and methods: Male rats (Sprague Dawley) (n=84) underwent abdominal wall surgery and suturing. Rats were randomized in groups according to evaluation time (2, 4, 6, 8, 24 hours and 7 days) with corresponding control groups. Controls received saline (0.9%) and the experimental groups received PL/PG on the surgery site. tPA, PAI-1, IL-6 and active TGFb1 were analyzed at given time points postoperatively in peritoneal lavage. Adhesions were evaluated after seven days. Significant differences were considered to be p<0.05. Results: At a few individual time points small differences were seen between the groups (control and experiment) comparing levels of tPA, PAI-1, IL-6 and active TGFb1. When comparing levels of substances from all time points no statistical differences were seen between the groups as a total. Adhesions were significantly decreased on day 7, p=0.002. Conclusion: Despite significant reduction in adhesions PL/PG administered intraperitoneally as an anti-adhesion agent locally on surgically traumatized area does not seem to affect the normal process of peritoneal healing.

Study of Synthesis and Properties on Poly L-Glutamate Initiated by Aniline

With L-glutamic acid as raw materials, N-carboxy-L-glutamic acid-benzyl-anhydride (BLG-NCA) was synthesized by triphosgene. With aniline as the initiator in the anhydrous dichloromethane, ring-opening polymerization of BLG-NCA was initiated, poly-l-glutamic acid benzyl ester was obtained. The amount of initiators, Molar ratio of monomer/initiators, reaction temperature and reaction time to the effect of poly-l-glutamic acid benzyl ester in the ring-opening polymerization of BLG-NCA were studied. Through infrared spectroscopy, gel permeation chromatography (GPC) analysis and characteristic viscosity method, polymerization product was characterized and analysized. Results showed that in dichloromethane, ring-opening polymerization of BLG-NCA could be initiated by aniline, which expands greater scope of the initiators of poly-l-glutamate to aromatic compounds, and the introduction of the active group provided by aromatic compounds make it possible for the advanced development of block copolymer with poly-l-glutamic acid as a basic unit.

Protection of the liver against CCl4-induced injury by intramuscular electrotransfer of a kallistatin-encoding plasmid

To investigate the effect of transgenic expression of kallistatin (Kal) on carbon tetrachloride (CCl(4))-induced liver injury by intramuscular (im) electrotransfer of a Kal-encoding plasmid formulated with poly-L-glutamate (PLG). The pKal plasmid encoding Kal gene was formulated with PLG and electrotransferred into mice skeletal muscle before the administration of CCl4. The expression level of Kal was measured. The serum biomarker levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), malonyldialdehyde (MDA), and tumor necrosis factor (TNF)-α were monitored. The extent of CCl4-induced liver injury was analyzed histopathologically. The transgene of Kal was sufficiently expressed after an im injection of plasmid formulated with PLG followed by electroporation. In the Kal gene-transferred mice, protection against CCl4-induced liver injury was reflected by significantly decreased serum ALT, AST, MDA and TNF-α levels compared to those in control mice (P<0.01 to 0.05 in a dose-dependent manner). Histological observations also revealed that hepatocyte necrosis, hemorrhage, vacuolar change and hydropic degeneration were apparent in mice after CCl4 administration. In contrast, the damage was markedly attenuated in the Kal gene-transferred mice. The expression of hepatic fibrogenesis marker transforming growth factor-β1 was also reduced in the pKal transferred mice. Intramuscular electrotransfer of plasmid pKal which was formulated with PLG significantly alleviated the CCl4-induced oxidative stress and inflammatory response, and reduced the liver damage in a mouse model.

Toxicity and Dose Response of Intra-Abdominally Administered Poly-L-α-Lysine and Poly-L-Glutamate for Postoperative Adhesion Protection

Background/Aims: Two differently charged polypeptides, poly-L-lysine (PL) and poly-L-glutamate (PG), have previously been shown to reduce postoperative intra-abdominal adhesions. This study aims to investigate the possible toxic effects and to establish a lowest effective antiadhesive dose. Methods: 152 mice were investigated with a well-known adhesion model and given different concentrations of the two differently charged polypeptides as well as only the cationic PL. Results: For the first time, a probable toxic level of PL given intraperitoneally (40 mg/kg) and the lowest significant concentration of PL and PG for antiadhesive purposes (1.6 mg/kg) could be established. Conclusion: The gap between the possible toxicity level of PL and the lowest efficient antiadhesive dose is probably too narrow, and the shape and charge of PL warrant continuous research for another polycation in the concept of differently charged polypeptides used as antiadhesive agents.

Review on Medusa®:a polymer-based sustained release technology for protein and peptide drugs

The polymer-based Medusa® system (Flamel Technologies) has been designed for slow release of therapeutic proteins and peptides. The Medusa II consists of a poly l-glutamate backbone grafted with hydrophobic α-tocopherol molecules, creating a colloidal suspension of nanoparticles (10 – 50 nm) in water. The sustained drug release is based on reversible drug interactions with hydrophobic nanodomains within the nanoparticles. In vivo, it is suggested that the therapeutic protein is displaced by endogenous proteins present in physiological fluids, leading to a slow drug release. The peak concentration is dramatically decreased and the protein release substantially extended. The Medusa technology has been applied to subcutaneous injection for several therapeutic proteins, such as IL-2 and IFN-α2b, in animal models (rats, dogs, monkeys) and clinical trials in renal cancer (IL-2) and hepatitis C (IFN-α2b) patients.

Preparation of Dendrimer SAM on Au Substrate and Adsorption/Desorption of Poly-&lt;I&gt;L&lt;/I&gt;-glutamate on the SAM

A self-assembled monolayer (SAM) of a dendrimer has been fabricated by covalent bonding of amine-terminated dendrimers to 3-mercaptopropionic acid SAM on an Au substrate, where an amide bond was formed between the amine and the carboxylic acid using amide-coupling reagents. Each reaction step was inspected by transmission surface-enhanced infrared absorption spectroscopy. Reaction was affected by solvents of the coupling reagents and concentrations of the coupling reagents and dendrimers. The pH-dependent adsorption/desorption behavior of sodium poly-L-glutamate (PGA) has been examined on the dendrimer SAM prepared. The adsorbed amount was abundant at acid pH below the pK(a) of carboxylate, while desorption from PGA-adsorbed dendrimer SAM prepared at acid pH proceeded as the pH was raised. The reaction was reversible and reproducible within 30 min under controlled pH, although the carboxylic acid species adsorbed at pH 4.2 was deprotonated to a carboxylate species after more than 30 min of desorption at pH 9.4.

Definition of the fine specificity of the monoclonal antibody 81D4: its reactivity with lysine and polyamine isopeptide cross-links

The 81D1C2 monoclonal antibody (Mab) directed against the N ɛ-(γ- l-glutamyl)- l-lysine isopeptide was found to cross-react on Enzyme Immuno Assay (EIA) with acylated lysines. Using a differential screening EIA procedure, a new Mab 81D4 was selected, which did not cross-react with acylated lysines but exhibited strong reactivity with N ɛ-(γ- l-glutamyl)- l-lysine formed by covalently coupling the γ-carboxyl of N αCBZ OtBu glutamic acid to Ɛ-NH 2 derivatized microtiter plates. When N ɛ-(γ- l-glutamyl)- l-lysine isopeptides were generated on γ-carboxyl derivatized plates, only lysine isopeptides with blocked α-amines were reactive, regardless of whether the bond formed by the amine blocking agent was a carbamate with carbobenzyloxychloride or an amide with acetic anhydride. The 81D4 Mab showed little or no affinity for free N ɛ-(γ- l-glutamyl)- l-lysine (IC 50>5 mM), for N 1 or N 4 mono(γ-Poly l-glutamyl)putrescine, and for N 1 mono(γ-Poly l-glutamyl)spermidine (IC 50>5 mM). However, when these same isopeptides were synthesized as cross-links between two protein chains— N ɛ-(γ- l-glutamyl)- l-lysine between Poly l-glutamate and Poly l-lysine; N 1 N 4- bis(γ-Poly l-glutamyl)putrescine, N 1 N 8- bis(γ-Poly l-glutamyl)spermidine between Poly- l-glutamate chains—very good reactivity was observed (IC 50 400 μM for lysine; 80 μM for putrescine and spermidine). In addition to the chemically synthesized isopeptide cross-links that were recognized by this Mab, the naturally occurring N ɛ-(ã- l-glutamyl)- l-lysine isopeptide cross-links in d-dimer, which are formed by the action of plasma transglutaminase (Factor XIII) on fibrin, were also detected on immunoblots using 81D4 as the primary antibody.

Electrochemical study of ion channel behavior in incorporated poly L-glutamate bilayer lipid membranes.

The lipid layer membranes were fabricated on the glassy carbon electrode (GC) and demonstrated to be bilayer lipid membranes by impedance spectroscopy. The formation of incorporated poly L-glutamate bilayer lipid membrane was achieved. The ion channel behavior of the incorporated poly L-glutamate membrane was determined. When the stimulus calcium cations were added into the electrolyte, the ion channel was opened immediately and exhibited distinct channel current. Otherwise, the ion channel was closed. The cyclic voltammogram at the GC electrode coated with incorporated poly L-glutamate DMPC film response to calcium ion is very fast compared with that at the GC electrode coated only with DMPC film. Ion channel current is not dependent on the time but on the concentration of calcium. The mechanism of the ion channel formation was investigated.

Binding-induced conformational transition of sodium poly-L-glutamate by iron(III) complex ions in aqueous solution

The binding of pseudo-octahedral trans- and cis-FeIII complex ions by sodium poly-L-glutamate (PLG) and dextransulphate (DS) in aqueous solution at about pH 7 has been studied. Equilibrium dialysis and “phase-separation” data show that the affinity for the complex counterions by both polyelectrolytes follows the order trans-[Fe(tetpy)(OH)2]+ > cis-[Fe(pmen)(OH)2]+. Evidence of a specific site binding, leading to a marked “renaturation” effect on the charged polypeptide, is produced in the case of the trans-FeIII-quaterpyridine compound. The binding isotherm of the trans-complex + PLG system and the circular dichroism patterns of the polypeptide as a function of the bound-trans-complex to polymer-residue ratio were successfully treated by a two-state model for the polyelectrolyte and a preferential association of the complex ions to the helical conformation of the polymeric matrix. All these features are examined in the light of the structural characteristics of the interacting species. The implications of the different stereochemistry of the other complex ions studied on the binding process with PLG are also discussed.