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Peptide dendrimers have a broad application in biomedical science due to their biocompatibility, diversity, and multifunctionality, but the precision synthesis of high-molecule weight peptide dendrimers remains challenging. We here report the facile and liquid-phase synthesis of molecular level precision and amino-acid built-in polylysine (PLL) dendrimers with molecular weights as high as ∼60 kDa. Three types of polyhedral oligosilsesquioxane (POSS)-cored PLL dendrimers with phenylalanine, tyrosine, or histidine as building blocks were synthesized. The precise structures of the dendrimers were confirmed by MALDI-TOF MS, GPC, and 1 H NMR spectroscopy. The interior functionalized peptide dendrimers improved the encapsulation capability of SN38 and sustained the release profiles. Enhanced molecular interactions between the peptide dendrimers and drugs were explored by both NMR experiments and computer simulations. The peptide dendrimer/SN38 formulations showed potent antitumor activity against multiple cancer cell lines. We believe that this strategy can be applied to the synthesis of tailor-made functional peptide dendrimers for drug-specific delivery and other diverse biomedical applications.

Developing strategies to optimize double-stranded RNA (dsRNA) delivery remains a significant challenge in improving RNA interference (RNAi) in insects. Nanoformulations may provide an avenue for the safe and effective delivery of dsRNA. We investigated nanoparticle-mediated gene silencing using biodegradable polymers, poly-l-lysine (PLL), and polyphenol (-)-epigallocatechin gallate (EGCG) for dsRNA delivery into Spodoptera frugiperda (Sf9) cells. Negatively charged cores were formed by EGCG and dsRNA complexes, and PLL was used to encapsulate the cores. The nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), and energy-dispersive spectrometry (EDS) analysis. The stability of the nanoparticles was assessed by incubating them in nuclease-containing Sf9 cell conditioned media. The effectiveness of the nanoparticles was investigated in Sf9 cells stably expressing the luciferase gene. The results revealed that the nanoparticles formed were small and spherical. The PLL/EGCG/dsRNA nanoparticles exhibited better stability compared to that of PLL/dsRNA or naked dsRNA. Nanoparticles prepared with dsRNA targeting the luciferase gene induced an efficient knockdown (66.7%) of the target gene. In Sf9 cells, nanoparticles prepared with Cy3- or CyPHer-5E-labeled dsRNA showed higher cellular uptake and endosomal escape, respectively, than the naked dsRNA. The improvement in uptake and cytosolic delivery may have helped to increase the knockdown efficiency. In Sf9 cells, the nanoparticles prepared with dsRNA targeting the inhibitor of apoptosis gene induced apoptosis by knocking down its expression. In conclusion, we demonstrate that PLL/EGCG/dsRNA nanoparticles are stable, highly efficient, and effective in dsRNA delivery and knockdown of the target gene.

The limited regenerative capacity of the CNS poses formidable challenges to the repair of spinal cord injury (SCI). Two key barriers to repair are (i) the physical gap left by the injury, and (ii) the inhibitory milieu surrounding the injury, the glial scar. Biomaterial implantation into the injury site can fill the cavity, provide a substrate for cell migration, and potentially attenuate the glial scar. We investigated the biological viability of a biocompatible and biodegradable poly-ε-lysine based biomaterial, Proliferate®, in low and high cross-linked forms and when coated with IKVAV peptide, for SCI implantation. We demonstrate altered astrocyte morphology and nestin expression on Proliferate® compared to conventional glass cell coverslips suggesting a less reactive phenotype. Moreover Proliferate® supported myelination in vitro, with myelination observed sooner on IKVAV-coated constructs compared with uncoated Proliferate®, and delayed overall compared with maintenance on glass coverslips. For in vivo implantation, parallel-aligned channels were fabricated into Proliferate® to provide cell guidance cues. Extensive vascularisation and cellular infiltration were observed in constructs implanted in vivo, along with an astrocyte border and microglial response. Axonal ingrowth was observed at the construct border and inside implants in intact channels. We conclude that Proliferate® is a promising biomaterial for implantation following SCI.

Introduction: Advanced tumor-targeted theranostic nanoparticles play a key role in tumor diagnosis and treatment research. In this study, we developed a multifunctional theranostic platform based on an amphiphilic hyaluronan/poly-( N -ε-carbobenzyloxy-L-lysine) derivative (HA- g -PZLL), superparamagnetic iron oxide (SPIO) and aggregation-induced emission (AIE) nanoparticles for tumor-targeted magnetic resonance (MR) and fluorescence (FL) dual-modal image-guided photodynamic therapy (PDT)., Materials and Methods: The amphiphilic hyaluronan acid (HA) derivative HA- g -PZLL was synthesized by grafting hydrophobic poly-( N -ε-carbobenzyloxy-L-lysine) (PZLL) blocks onto hyaluronic acid by a click conjugation reaction. The obtained HA- g -PZLLs self-assembled into nanoparticles in the presence of AIE molecules and SPIO nanoparticles to produce tumor-targeted theranostic nanoparticles (SPIO/AIE@HA- g -PZLLs) with MR/FL dual-modal imaging ability. Cellular uptake of the theranostic nanoparticles was traced by confocal laser scanning microscopy (CLSM), flow cytometry and Prussian blue staining. The intracellular reactive oxygen species (ROS) generation characteristics of the theranostic nanoparticles were evaluated with CLSM and flow cytometry. The effect of PDT was evaluated by cytotoxicity assay. The dual-mode imaging ability of the nanoparticles was evaluated by a real-time near-infrared fluorescence imaging system and magnetic resonance imaging scanning., Results: The resulting theranostic nanoparticles not only emit red fluorescence for high-quality intracellular tracing but also effectively produce singlet oxygen for photodynamic tumor therapy. In vitro cytotoxicity experiments showed that these theranostic nanoparticles can be efficiently taken up and are mainly present in the cytoplasm of HepG2 cells. After internalization, these theranostic nanoparticles showed serious cytotoxicity to the growth of HepG2 cells after white light irradiation., Discussion: This work provides a simple method for the preparation of theranostic nanoparticles with AIE characteristics and MR contrast enhancement, and serves as a dual-modal imaging platform for image-guided tumor PDT., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Yang et al.)

Bioreducible cationic polymers have gained considerable attention in gene delivery due to their low cytotoxicity and high efficiency. In the present work, we reported a cationic polymer, poly(disulfide-l-lysine)-g-agmatine (denoted as SSL-AG), and evaluated its ability to transfer pEGFP-ZNF580 plasmid (pZNF580) into human umbilical vein endothelial cells (HUVECs). This SSL-AG polymeric carrier efficiently condensed pZNF580 into positively charged particles (<200 nm) through electrostatic interaction. This carrier also exhibited excellent buffering capacity in the physiological environment, good pDNA protection against enzymatic degradation and rapid pDNA release in a highly reducing environment mainly because of the responsive cleavage of disulfide bonds in the polymer backbone. The hemolysis assay and in vitro cytotoxicity assay suggested that the SSL-AG carrier and corresponding gene complexes possessed both good hemocompatibility and great cell viability in HUVECs. The cellular uptake of the SSL-AG/Cy5-oligonucleotide group was 3.6 times that of the poly(l-lysine)/Cy5-oligonucleotide group, and its mean fluorescence intensity value was even higher than that of the PEI 25 kDa/Cy5-oligonucleotide group. Further, the intracellular trafficking results demonstrated that the SSL-AG/Cy5-oligonucleotide complexes exhibited a high nucleus co-localization rate (CLR) value (36.0 ± 2.8%, 3.4 times that of the poly (l-lysine)/Cy5-oligonucleotide group, 1.6 times that of the poly(disulfide-l-lysine)-g-butylenediamine/Cy5-oligonucleotide group) at 24 h, while the endo/lysosomal CLR value was relatively low. This suggested that SSL-AG successfully delivered plasmid into HUVECs with high cellular uptake, rapid endosomal escape and efficient nuclear accumulation owing to the structural advantages of the bioreducible and agmatine groups. In vitro transfection assay also verified the enhanced transfection efficiency in the SSL-AG/pZNF580 group. Furthermore, the results of CCK-8, cell migration and in vitro/vivo angiogenesis assays revealed that pZNF580 delivered by SSL-AG could effectively enhance the proliferation, migration and vascularization of HUVECs. In a word, the SSL-AG polymer has great potential as a safe and efficient gene carrier for gene therapy.

Penicillin G acylase (PGA) from Providencia rettgeri PX04 (PrPGA) was utilized to synthesize β-lactam antibiotics. Poly-lysine supported cross-linked enzyme aggregates (PL-CLEAs) were prepared using PGA. Addition of poly-lysine significantly increased retention of PGA activity in CLEAs, with a decrease in the synthesis/hydrolysis (S/H) ratio. PL-CLEAs with 0.56 mg/mL poly-lysine retained 83% of free PGA activity, and displayed a higher S/H ratio than that of the free enzyme. Both PL-CLEAs and CLEAs exhibited high pH and thermal stabilities. PL-CLEAs possessed the best stability profile, and the lowest α value [(k cat /K m ) Ps /(k cat /K m ) AD ], and was most effective at amoxicillin synthesis. A >94% yield of amoxicillin was achieved using a D-HPGME/6-APA ratio of 1.2:1 (240 mM, 200 mM), with fed-batch addition of D-HPGME. PL-CLEAs displayed excellent operational stability during amoxicillin synthesis. Over 97% of initial conversion was retained after twenty rounds of catalysis. PL-CLEAs exhibited greater potency than CLEAs in practical catalysis, permitting a higher concentration of reactants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Stimuli-responsive biomaterials supply a promising solution to adapt to the complex physiological environment for different biomedical applications. In this study, a dynamic UV-triggered pH-responsive biosurface was constructed on titania nanotubes (TNTs) by loading photoacid generators, diphenyliodonium chloride, into the nanotubes, and grafting 2,3-dimethyl maleic anhydride (DMMA)-modified hyperbranched poly(l-lysine) (HBPLL) onto the surface. The local acidity was dramatically enhanced by UV irradiation for only 30 s, leading to the dissociation of DMMA and thereby the transformation of surface chemistry from negatively charged caboxyl groups to positively charged amino groups. The TNTs-HBPLL-DMMA substrate could better promote proliferation and spreading of rat bone mesenchymal stem cells (rBMSCs) after UV irradiation. The osteogenic differentiation of rBMSCs was enhanced because of the charge reversal in combination with the titania-based substrates.

We developed a tumor-targeted contrast agent based on linear polylysine (PLL) by conjugating a small molecular imaging agent, fluorescent molecule and targeting agent amino phenylboronic acid onto the amino groups of polylysine, which can specifically target monosaccharide sialic acid residues overexpressing on the surface of tumor cell membranes. Further, 3,4,5,6-Tetrahydrophthalic anhydride (DCA) was attached to the free amino groups of the polylysine to change to a negative charge at physiology pH to lower the cytotoxicity, but it soon regenerated to a positive charge again once reaching the acidic intratumoral environment and therefore increased cell uptake. Laser confocal microscopy images showed that most of the polymeric contrast agents were bound to the cancer cell membrane. Moreover, the tumor targeting contrast agent showed the same magnetic resonance imaging (MRI) contrasting performance in vitro as the small molecule contrast agent used in clinic, which made it a promising tumor-targeting polymeric contrast agent for cancer diagnosis.

Background: Engineered inorganic nanoparticles (NPs) are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. We show that by changing the coating of magnetic iron oxide NPs using poly-L-lysine (PLL) polymer the colloidal stability of the dispersion is improved in aqueous solutions including water, phosphate buffered saline (PBS), PBS with 10% fetal bovine serum (FBS) and cell culture medium, and the internalization of the NPs toward living mammalian cells is profoundly affected., Methods: A multifunctional magnetic NP is designed to perform a near-infrared (NIR)-responsive remote control photothermal ablation for the treatment of breast cancer. In contrast to the previously reported studies of gold (Au) magnetic (Fe 3 O 4 ) core-shell NPs, a Janus-like nanostructure is synthesized with Fe 3 O 4 NPs decorated with Au resulting in an approximate size of 60 nm mean diameter. The surface of trisoctahedral Au-Fe 3 O 4 NPs was coated with a positively charged polymer, PLL to deliver the NPs inside cells. The PLL-Au-Fe 3 O 4 NPs were characterized by transmission electron microscopy (TEM), XRD, FT-IR and dynamic light scattering (DLS). The unique properties of both Au surface plasmon resonance and superparamagnetic moment result in a multimodal platform for use as a nanothermal ablator and also as a magnetic resonance imaging (MRI) contrast agent, respectively. Taking advantage of the photothermal therapy, PLL-Au-Fe 3 O 4 NPs were incubated with BT-474 and MDA-MB-231 breast cancer cells, investigated for the cytotoxicity and intracellular uptake, and remotely triggered by a NIR laser of ~ 808 nm (1 W/cm 2 for 10 min)., Results: The PLL coating increased the colloidal stability and robustness of Au-Fe 3 O 4 NPs (PLL-Au-Fe 3 O 4 ) in biological media including cell culture medium, PBS and PBS with 10% fetal bovine serum. It is revealed that no significant (< 10%) cytotoxicity was induced by PLL-Au-Fe 3 O 4 NPs itself in BT-474 and MDA-MB-231 cells at concentrations up to 100 μg/ml. Brightfield microscopy, fluorescence microscopy and TEM showed significant uptake of PLL-Au-Fe 3 O 4 NPs by BT-474 and MDA-MB-231 cells. The cells exhibited 40 and 60% inhibition in BT-474 and MDA-MB-231 cell growth, respectively following the internalized NPs were triggered by a photothermal laser using 100 μg/ml PLL-Au-Fe 3 O 4 NPs. The control cells treated with NPs but without laser showed < 10% cell death compared to no laser treatment control CONCLUSION: Combined together, the results demonstrate a new polymer gold superparamagnetic nanostructure that integrates both diagnostics function and photothermal ablation of tumors into a single multimodal nanoplatform exhibiting a significant cancer cell death.

High transfection efficiency and low cytotoxicity are the two key factors to be considered in the design of gene carriers. Herein, a novel and versatile gene carrier (PLL-RT) was prepared by introducing "molecular string" RT (i.e., p-toluylsulfonyl arginine) onto the polylysine backbone. The introduction of RT string contributed to the formation of multiple interactions between the polycationic gene carriers and cell membrane or DNA, as well as adopting α-helix conformation, all of which would be beneficial to enhance the gene transfection. In addition, RT string grafted onto other polycations such as hyperbranced PEI25k and dendrimer PAMAM could also acquire improved transfection efficiency and low cytotoxicity. Moreover, PLL-RT presented significant tumor inhibition effect in vivo. This work provided an effective strategy for constructing novel gene carriers with high transfection and low cytotoxicity.

Background: Lumbrokinase (LK) is an enzyme complex with antithrombotic, antioxidant, antitumor, and immunomodulatory effects. It has been extensively studied and used in clinical anti-tumor therapy. However, its half-life is short, its bioavailability is low, and its toxicity and side effects are great, which greatly limit its clinical application. Therefore, LK is often combined with other drugs (such as immune agents, hormones, or Chinese herbal medicine) to reduce its dosage and side effects and to improve its anti-tumor effects., Methods and Results: Here, we described an LK/paclitaxel (PTX) nanocarrier based on poly(ethylene glycol)- b -(poly(ethylenediamine l-glutamate)- g -poly(ε-benzyoxycarbonyl-l-lysine)- r -poly(l-lysine)) (PEG- b -(PELG- g -(PZLL- r -PLL))). In the present study, LK and PTX were loaded by electrostatic and/or hydrophobic effects under mild conditions, thereby increasing the half-life and bioavailability of the drugs via the sustained release and enhancement of tumor site enrichment by the LK/PTX/PEG- b -(PELG- g -(PZLL- r -PLL)) complex through passive targeting. In this study, using bladder cancer cells (J82 cells) and rat bladder cancer model as the object, the structure of the nanocarrier, the relationship between drugs composition and antitumor properties were systematically studied., Conclusion: We propose that the block copolymer PEG- b -(PELG- g -(PZLL- r -PLL)) may function as a potent nanocarrier for augmenting anti-bladder cancer pharmacotherapy, with unprecedented clinical benefits., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A simple, noncovalent modification strategy was proposed to synthesize poly-l-lysine-black phosphorus (pLL-BP) hybrid. BP nanoflakes were prepared with a water-phase exfoliation method. pLL can adhere to the surface of BP via hydrophobic interaction between butyl chains of pLL and the BP surface as well as the electrostatic interaction between the protonated amino groups on pLL and the negative charge on deprotonated P x O y groups remaining on BP. The as-synthesized pLL-BP hybrid turns out to be an ideal matrix for hemoglobin immobilization and direct electron transfer. Good conductivity and biocompatibility of BP maintain the native structure and the bioactivity of hemoglobin (Hb), facilitating the direct electron transfer between the electroactive center of Hb and electrode. The rate constant ( k ET ) for direct electron transfer of Hb@pLL-BP is calculated to be 11.24 s -1 . The constructed Hb-pLL-BP based enzymatic electrochemical biosensor displays excellent catalytic activity toward the reduction of oxygen and hydrogen peroxide. The electrochemical response toward H 2 O 2 exhibits a linear dependence on hydrogen peroxide concentration ranging between 10 μM and 700 μM. The results demonstrate that the pLL-BP hybrid can act as a biocompatible building block for the construction of novel biofuel cells, bioelectronics, and biosensors.

The purpose of this research was to evaluate the protective effects of insulin-loaded poly(ethylene glycol)-b-poly((2-aminoethyl-l-glutamate)-g-poly(l-lysine)) (PEG-b-P(ELG-g-PLL)) on renal ischemia/reperfusion (I/R) injury in rats with diabetes mellitus. Rats were preconditioned with free insulin or insulin/PEG-b-P(ELG-g-PLL) polyplexes, then subjected to renal I/R. The blood and kidneys were then harvested, Glucose uptake rate, glucose transporter 4 (GULT4) mRNA level, cell membrane GULT4 content and GULT4 expression were measured, the level of serum creatinine and blood urea nitrogen were determined, the activity of superoxide dismutase and inducible nitric oxide synthase, the content of malondialdehyde and nitric oxide, reactive oxygen species (ROS) production and nuclear factor κB (NF-κB) mRNA level, Bcl-2 assaciated x protein (Bax) mRNA and B cell lymphoma/lewkmia-2 (Bcl-2) mRNA level, and the expression of protein 47kDa phagocyte oxidase (p47phox) in renal tissues were measured. Insulin preconditioning improved the recovery of renal function, reduced oxidative stress injury, restored nitroso-redox balance and downregulated the expression of p47phox induced by renal I/R injury, while the application of block copolymer PEG-b-P(ELG-g-PLL) as an insulin nanocarrier significantly enhanced the protective effect of insulin. Block copolymer PEG-b-P(ELG-g-PLL) could be used as a potential nanocarrier for insulin with sustained release and enhanced bioavailability., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Cationic macromolecular carriers can be effective carriers for small molecular compounds, drugs, epitopes, or nucleic acids. Polylysine-based polymeric branched polypeptides have been systematically studied on the level of cells and organisms as well. In the present study, we report our findings on the cellular uptake characteristics of nine structurally related polylysine-based polypeptides with cationic side chains composed of (i) single amino acid (poly[Lys(X i )], X i K) or (ii) oligo[dl-alanine] (poly[Lys(dl-Ala m )], AK) or (iii) oligo[dl-alanine] with an additional amino acid (X) at the terminal position (poly[Lys(X i -dl-Ala m )] (XAK)) or (iv) at the position next to the polylysine backbone (poly[Lys(dl-Ala m -X i )] (AXK)). In vitro cytotoxicity and cellular uptake were characterized on HT-29 human colon carcinoma and HepG2 human hepatocarcinoma cell lines. Data indicate that the polycationic polypeptides studied are essentially nontoxic in the concentration range studied, and their uptake is very much dependent on the side chain structure (length, identity of amino acid X, and distance between the terminal positive charges) and also on the cell lines. Our findings in uptake inhibition studies suggest that predominantly macropinocytosis and caveole/lipid raft mediated endocytosis are involved. The efficacy of their internalization is markedly influenced by the hydrophobicity and charge properties of the amino acid X. Interestingly, the uptake properties of the these polypeptides show certain similarities to the entry pathways of several cell penetrating peptides.

Brain ischemia/reperfusion (I/R) injury (BI/RI) is a leading cause of death and disability worldwide. However, the outcome of pharmacotherapy for BI/RI remains unsatisfactory. Innovative approaches for enhancing drug sensitivity and recovering neuronal activity in BI/RI treatment are urgently needed. The purpose of this study was to evaluate the protective effects of tumor necrosis factor (TNF)-α-loaded poly(ethylene glycol)- b -(poly(ethylenediamine L-glutamate)- g -poly(L-lysine)) (TNF-α/PEG- b -(PELG- g -PLL)) nanoparticles on BI/RI. The particle size of PEG- b -(PELG- g -PLL) and the loading and release rates of TNF-α were determined. The nanoparticle cytotoxicity was evaluated in vitro using rat cortical neurons. Sprague Dawley rats were preconditioned with free TNF-α or TNF-α/PEG- b -(PELG- g -PLL) polyplexes and then subjected to 2 hours ischemia and 22 hours reperfusion. Brain edema was assessed using the brain edema ratio, and the antioxidative activity was assessed by measuring the superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content in the brain tissue. We further estimated the inflammatory activity and apoptosis level by determining the levels of interleukin-4 (IL-4), IL-6, IL-8, IL-10, and nitric oxide (NO), as well as the expression of glial fibrillary acidic protein (GFAP), intercellular adhesion molecule-1 (ICAM-1), and cysteine aspartase-3 (caspase-3), in the brain tissue. We provide evidence that TNF-α preconditioning attenuated the oxidative stress injury, the inflammatory activity, and the apoptosis level in I/R-induced cerebral injury, while the application of block copolymer PEG- b -(PELG- g -PLL) as a potential TNF-α nanocarrier with sustained release significantly enhanced the bioavailability of TNF-α. We propose that the block copolymer PEG- b -(PELG- g -PLL) may function as a potent nanocarrier for augmenting BI/RI pharmacotherapy, with unprecedented clinical benefits. Further studies are needed to better clarify the underlying mechanisms., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

A new dendrimer probe was designed for bone imaging. Bidentate iminodiacetate groups were introduced to the probe to obtain strong bind to bones. The assembled dendrimeric probe, with four iminodiacetate moieties and a fluorescent tag, displayed good selectivity to hydroxyapatite, calcium oxalate and calcium phosphate salts. In mice, the probe offered vivid skeletal details after intravenous delivery., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Unlabelled: Vascular endothelial growth factor (VEGF) is the growth factor responsible for the triggering of angiogenesis, the process of blood vessel formation supporting the long-term viability of any repaired or regenerated tissue. As the growth factor is effective only when concentration gradients are generated, new shuttles need to be developed that ensure both the control of gradients at the site of tissue repair and the release of VEGF at physiological levels. Magnetic hyperthermia is the production of heat induced by magnetic materials through their exposure to an external oscillating magnetic field. In this paper, magnetic nanoparticles capable of generating controllable hyperthermia were functionalised with hyperbranched poly(epsilon-lysine) peptides integrating in their core parallel thermoresponsive elastin-like peptide sequences and presenting an uppermost branching generation tethered by the zwitterionic amino acid carboxybetaine. The results show that these functionalised magnetic nanoparticles avidly bind VEGF and release it only upon generation of mild-hyperthermic pulses generated by oscillating magnetic filed. The VEGF release occurred in a temperature range at which the elastin-like peptides collapse. It is proposed that, through the application of an external magnetic field, these magnetic carriers could generated gradients of VEGF in vivo and allow its tuned delivery in a number of clinical applications., Statement of Significance: The present paper for the first time reveals the possibility to control the delivery of VEGF through mild hyperthermia stimuli generated by a oscillating magnetic field. To this purpose, magnetic nanoparticles of high size homogeneity and coated with a thin coating of poly(acrylic acid) were functionalised with a novel class of poly(epsilon lysine) dendrimers integrating in their structure a thermoresponsive amino acid sequence mimicking elastin and exposing at high density a zwitterionic modified amino acid, the carboxybetaine, known to be able to bind macromolecules. Physicochemical and biochemical characterisation elegantly show the link between the thermal properties of the nanoparticles and of the dendrimer change of conformation and how this enable the release of VEGF at temperature values compatible with the growth factor stability., (Copyright © 2016. Published by Elsevier Ltd.)

The aim of this study was to observe the therapeutic effect of insulin-loaded linear poly(ethylene glycol)-brush-like poly(l-lysine) block copolymer poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)) (PEG-b-(PELG-g-PLL) on renal ischemia/reperfusion-induced lung injury through downregulating hypoxia-inducible factor (HIF) as compared to free insulin. Sprague Dawley rats were pretreated with 30 U/kg insulin or insulin/PEG-b-(PELG-g-PLL) complex, and then subjected to 45 minutes of ischemia and 24 hours of reperfusion. The blood and lungs were collected, the level of serum creatinine and blood urea nitrogen were measured, and the dry/wet lung ratios, the activity of superoxide dismutase and myeloperoxidase, the content of methane dicarboxylic aldehyde and tumor necrosis factor-α, and the expression of HIF-1α and vascular endothelial growth factor (VEGF) were measured in pulmonary tissues. Both insulin and insulin/PEG-b-(PELG-g-PLL) preconditioning improved the recovery of renal function, reduced pulmonary oxidative stress injury, restrained inflammatory damage, and downregulated the expression of HIF-1α and VEGF as compared to ischemia/reperfusion group, while insulin/PEG-b-(PELG-g-PLL) significantly improved this effect.

Peptide-decorated dendrimers (PDDs) are a class of spherical, regular, branched polymers that are modified by peptides covalently attached to their surface. PDDs have been used as protein mimetics, novel biomaterials, and in a wide range of biomedical applications. Since their design and development in the late eighties, poly-l-lysine has been a preferred core structure for PDDs. However, numerous recent innovations in polymer synthesis and ligation chemistry have re-energized the field and led to the emergence of well-defined peptide dendrimers with more diverse core structures and functions. This Minireview highlights the development of PDDs driven by significantly improved ligation chemistry incorporating structurally well-defined peptides and the emerging use of PDDs in imaging and drug development., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chlorin e6 is a tricarboxylic acid degradation product of chlorophyll a. Four chlorin e6 bis(amino acid) conjugates were regioselectively synthesized bearing two aspartate conjugates in the 13(1),17(3)- and 15(2),17(3)-positions, or at the 13(1),15(2)via an ethylene diamine linker. One additional conjugate bearing two different amino acids, lysine at 13(1)via an ethylene diamine linker and an aspartate at 15(2)via a β-alanine linker was also synthesized. The cytotoxicity and uptake of four di(amino acid) chlorin e6 conjugates were investigated in human HEp2 cells, and compared with chlorin e6. The most cytotoxic and most taken up conjugates were the zwitterionic 13(1),15(2)-disubstituted conjugates 28 and 33; these also localized in multiple organelles. In contrast, the anionic 13(1),17(3)- and 15(2),17(3)-di-aspartyl chlorin e6 conjugates 12 and 13 showed low dark cytoxicity and lower phototoxicity compared with chlorin e6.

Polylysine-b-p[HPMA] block copolymers containing a redox-responsive disulfide bond between both blocks are synthesized by RAFT polymerization of pentafluorphenyl-methacrylate with a macro-CTA from Nϵ-benzyloxycarbonyl (Cbz) protected polylysine (synthesized by NCA polymerization). This polylysine-b-p[PFMA] precursor block copolymer is converted to polylysine(Cbz)-b-p[HPMA] by postpolymerization modification with 2-hydroxypropylamine. After removal of the Cbz protecting group, cationic polylysine-b-p[HPMA] copolymers with a biosplittable disulfide moiety became available, which can be used as polymeric transfection vectors. These disulfide linked polylysine-S-S-b-p[HPMA] block copolymers show low cytotoxicity and increased transfection efficiencies (HEK-293T cells) compared to analogous blockcopolymers without disulfide group making them interesting for the transfection of sensitive immune cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Zwitterionic copolymers have exhibited high resistance to nonspecific protein adsorption and have wide applications in drug delivery systems. Herein, a pH-responsive poly(Lysine-alt-N,N'-bis(acryloyl) diaminohexane) was synthesized through the Michael addition polymerization between N, N'-bis(acryloyl) diaminohexane and lysine. Subsequently, nano micelles (NMs) were formed by self-assembly of the copolymer in an aqueous solution. The NMs showed a slightly negative charge in blood environment, but a positively charged surface in extracellular pH of tumor. This feature could be used to enhance permeability and retention effect, and reinforce tumor cell uptake. Vitro release studies revealed that the release of DOX from the DOX-loaded NMs was evidently faster at pH 5.0 than at pH 7.4. MTT assays revealed that NMs were nontoxic. Thus, these smart NMs were feasible candidates and could be potentially used in cancer chemotherapy.

Poly(L-lysine)s (PLLs), and related derivatives, have received considerable attention as nonviral vectors. High molecular weight PLLs (H-PLLs) are superior transfectants compared with low Mw PLLs (L-PLLs), but suggested to be more cytotoxic. Through a pan-integrated metabolomic approach using Seahorse XF technology, we studied the impact of PLL size on cellular bioenergetic processes in two human cell lines. In contrast to L-PLLs (1-5 kDa), H-PLLs (15-30 kDa) were more detrimental to both mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic activity resulting in considerable intracellular ATP depletion, thereby initiating necrotic-type cell death. The cellular differences to polycation sensitivity were further related to the mitochondrial state, where the impact was substantial on cells with hyperpolarized mitochondria. These medium-throughput approaches offer better opportunities for understanding inter-related intracellular and cell type-dependent processes instigating a bioenergetics crisis, thus, aiding selection (from available libraries) and improved design of safer biodegradable polycations for nucleic acid compaction and cell type-specific delivery.

The synthesis of triblock copolymers based on polysarcosine, poly-N-ε-t-butyloxycarbonyl-l-lysine, and poly-N-ε-t-trifluoroacetyl-l-lysine by ring-opening polymerization of the corresponding α-amino acid N-carboxyanhydrides (NCAs) is described. For the synthesis of N-ε-t-butyloxycarbonyl-l-lysine (lysine(Boc)) NCAs, an acid-free method using trimethylsilylchloride/triethylamine as hydrochloric acid (HCl) scavengers is presented. This approach enables the synthesis of lysine(Boc) NCA of high purity (melting point 138.3 °C) in high yields. For triblock copolypept(o)ides, the degree of polymerization (Xn ) of the polysarcosine block is varied between 200 and 600; poly-N-ε-t-butyloxycarbonyl-l-lysine and poly-N-ε-t-trifluoroacetyl-l-lysine blocks are designed to have a Xn in the range of 10-50. The polypeptide-polypeptoid hybrids (polypept(o)ides) can be synthesized with precise control of molecular weight, high end group integrity, and dispersities indices between 1.1 and 1.2. But more important, the use of tert-butyloxycarbonyl- and trifluoroacetyl-protecting groups allows the selective, orthogonal deprotection of both blocks, which enables further postpolymerization modification reactions in a block-selective manner. Therefore, the presented synthetic approach provides a versatile pathway to triblock copolypept(o)ides, in which functionalities can be separated in specific blocks., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new kind of biodegradable poly(urethane-urea)s based on poly-lysine oligomer used as the soft segment was synthesized and characterized. In vitro degradation behavior of poly (urethane-urea)s was investigated, and was assessed by (1)H NMR and mass loss. The results indicated that the peptide bonds in poly(urethane-urea)s were effectively cleaved in simulated pancreatic juice containing trypsin, while those in buffer solution without trypsin remained unaffected. The degradability was obviously improved by introducing poly-lysine oligomer into the main chain of poly(urethane-urea)s. The results of cells viability test indicated that the poly (urethane-urea)s showed a good biocompatibility on endothelial cells. The thermostability and hydrophilicity of poly(urethane-urea)s increased with increase in poly-lysine oligomer content.

For small interfering RNA (siRNA)-based cancer therapies, we report an actively-targeted and stabilized polyion complex micelle designed to improve tumor accumulation and cancer cell uptake of siRNA following systemic administration. Improvement in micelle stability was achieved using two stabilization mechanisms; covalent disulfide cross-linking and non-covalent hydrophobic interactions. The polymer component was designed to provide disulfide cross-linking and cancer cell-targeting cyclic RGD peptide ligands, while cholesterol-modified siRNA (Chol-siRNA) provided additional hydrophobic stabilization to the micelle structure. Dynamic light scattering confirmed formation of nano-sized disulfide cross-linked micelles (<50 nm in diameter) with a narrow size distribution. Improved stability of Chol-siRNA-loaded micelles (Chol-siRNA micelles) was demonstrated by resistance to both the dilution in serum-containing medium and counter polyion exchange with dextran sulfate, compared to control micelles prepared with Chol-free siRNA (Chol-free micelles). Improved stability resulted in prolonged blood circulation time of Chol-siRNA micelles compared to Chol-free micelles. Furthermore, introduction of cRGD ligands onto Chol-siRNA micelles significantly facilitated accumulation of siRNA in a subcutaneous cervical cancer model following systemic administration. Ultimately, systemically administered cRGD/Chol-siRNA micelles exhibited significant gene silencing activity in the tumor, presumably due to their active targeting ability combined with the enhanced stability through both hydrophobic interactions of cholesterol and disulfide cross-linking., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure-activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.

Well-defined amphiphilic polymers of the ABA and ABC type are synthesized, where A is poly(L-lysine hydrochloride) (PLL), B is poly(γ-benzyl-(d7) L-glutamate) (PBLG(-d7)), and C is poly(ethylene oxide) (PEO). The two polymers exhibit similar PBLG(-d7) composition, while in the ABC, the volume fraction of PEO block is higher than that of PLL. Both polymers form polymersomes in water. The polymersomes are loaded with doxorubicin or paclitaxel. It is found that in the ABC, due to asymmetry of the two hydrophilic blocks, PEO is always on the outer periphery and the dimensions of the vesicles are smaller. The release of the vesicles is temperature- and pH-dependent. In vivo toxicity tests of the empty vesicles show that they are not toxic. In vitro activity of the loaded vesicles against human pancreatic cancer cell lines reveals comparable activity to Myocet for the ABA loaded with doxorubicin, while lower activity is observed for the ABC., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Poly-L-lysine (PLL), which has been employed as a conductive polymer in the construction of some electrochemical sensors, can be prepared using L-lysine by cyclic voltammetry (CV) with a wide potential range. However, the presented explanation and description about its polymerization mechanism seems oversimplified because the self-reaction of electrode and the electrolysis of solvent at high potential are ignored. This work presents an intensive investigation on the relevant reactions during the process of PLL-polymerization using CV, X-ray photoelectron spectroscopy, Fourier transform-infrared spectroscopy, and electrochemical impedance spectroscopy. At a higher positive potential, the transfer from lysine molecules to cation radicals and the polymerization reaction on the glassy carbon electrode (GCE) could be achieved, accompanied by the activation of GCE, the formation of oxygen-containing functional groups, and the generation of oxygen derived from the oxidation of water. The adsorbed oxygen had a seriously negative effect on the formation of PLL unless it suffered reduction at a lower negative potential. The charge transfer through the electrochemical polymerized PLL film was seriously hindered by the immobilization of suspension cells due to the electrostatic interaction. The charge-transfer resistance difference (ΔR(ct)) was increased with the enhancement of the cell number (N(cells)) and the 1/ΔR(ct) value displayed a linear response with 1/N(cells) in the range of 5.0 × 10(2)-1.0 × 10(5) cells with a detection limit of 180 cells estimated at a signal-to-noise ratio of 3. A sensitive electrochemical sensor for the quantitative detection of suspension cells was developed.

The co-delivery of drug and gene has become the primary strategy in cancer and other disease therapy. To co-deliver hydrophobic drug and functional gene efficiently into tumor cells, a star-shaped copolymer (PP-PLLD-Arg) with a photochemical internalization effect consisting of a porphyrin (PP) core and arginine-functionalized poly(L-lysine) dendron (PLLD-Arg) arms has been designed, and used to co-deliver docetaxel (DOC) and MMP-9 shRNA plasmid for nasopharyngeal cancer therapy. It was found that PP-PLLD-Arg/MMP-9 nanocomplex showed the photo-enhanced gene transfection efficiency in vitro, and could mediate a significant reduce of MMP-9 protein expression in HNE-1 cells. For co-delivery analysis, the obtained PP-PLLD-Arg/DOC/MMP-9 complexes could induce a more significant apoptosis than DOC or MMP-9 used only, and decreased invasive capacity of HNE-1 cells. Moreover, the star-shaped copolymer exhibited better blood compatibility and lower cytotoxicity compared to PEI-25k in the hemolysis and MTT assays, and also showed a good biocompatibility in vivo. Therefore, PP-PLLD-Arg with suited irradiation is a promising non-toxic and photo-inducible effective drug and gene delivery strategy, which should be encouraged in tumor therapy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distribution in animals by enhancing the water proton signal intensity by the overhauser effect. In this study, we constructed a contrast agent by combining PROXYL groups that have nitroxyl radicals with PEG-modified dendritic poly(l-lysine) that accumulates in the tumor by enhanced permeability and retention (EPR) effect. Addition of the PROXYL groups at the PEG chains' termini on KG6 was advantageous in OMRI, because the ESR signal of the nitroxyl radical was maintained without decay caused by mobility restriction, even if the PROXYL groups were attached at 25 mol% on one molecule. After intramuscular injection of the molecule modified at 25 mol%, that is, PR25-PEG-KG6, a significant OMRI signal was observed at the injected site. However, no signal was detected in the tumor after intravenous injection of PR25-PEG-KG6 to a tumor-bearing mouse, although PR25-PEG-KG6 itself accumulated in the tumor. The reason was that the nitroxyl radicals were immediately reduced in the blood after the injection, suggesting that use of stable nitroxyl radicals will enable detection of tumors by OMRI after intravenous injection.

To deliver anti-cancer drugs to tumors, a hydrophobic cavity was prepared in the dendritic molecule, dendritic poly(L-lysine) of sixth generation (KG6), which was used as a drug carrier. The dendritic molecule was modified with polyethylene glycol (PEG)-linked hydrophobic penta-phenylalanine or penta-alanine. The hydrophobic cavity was formed between the KG6 and PEG chains. The penta-phenylalanine peptide was better in encapsulating doxorubicin (DOX) in the cavity compared with penta-alanine. The loaded DOX was slowly released from the cavity, and it depended on pH. After intravenous injection, the DOX-loaded dendrimers accumulated in the tumor by the enhanced permeability and retention effect, and showed significant suppression of tumor growth without loss of body weight. These results indicate that hydrophobic oligopeptides can be used for forming a hydrophobic cavity in a dendritic molecule for delivery of anti-cancer drugs to tumor sites.

A novel linear-dendron-like polyampholyte, poly(L-lysine)-b-D2-poly(L-glutamic acid) [PLL-b-D2-(PLGA)4], where D2 is the second generation of poly(amido amine), was prepared by hydrolyzing poly(ε-benzyloxycarbonyl-L-lysine)-b-D2-poly(γ-benzyl-L-glutamate) copolymer which was synthesized via a combination of ring-opening polymerization and click chemistry. The pH-responsive self-assembly behaviors of PLL-b-D2-(PLGA)4 were investigated in detail. It is found that PLL-b-D2-(PLGA)4 can self-assemble into PLGA-core aggregates at acidic pH and PLL-core aggregates at alkaline pH, which was accompanied with the coil-to-helix conformational transition of PLGA and PLL segments, respectively. The self-assembled aggregates with various morphologies, such as large compound micelles, worm-like micelles, large compound vesicles, simple vesicles, and rigid tubular structures have been obtained in "schizophrenic" aggregation process with simply increasing the solution pH. The hierarchical assembled fractal structures of PLL-b-D2-(PLGA)4 were observed during the solvent evaporation at high pH value.

Elongated micelles have many desirable characteristics for cancer-drug delivery, but they are difficult to obtain since amphiphilic polymers form such nanostructures only within narrow composition ranges depending on their own structures. Herein, we demonstrated a facile fabrication of different nanostructures via drug content-controlled self-assembly of amphiphilic linear-dendritic drug conjugates - using the number of the conjugated hydrophobic drug molecule camptothecin (CPT) to tailor the hydrophobicity of amphiphilic PEG-block-dendritic polylysine-CPT (PEG-xCPT) conjugates and thereby control their self-assembled nanostructures - nanospheres or nanorods of different diameters and lengths. The shape and size of the nanostructures were found to strongly affect their in vitro and in vivo properties, particularly the blood clearance kinetics, biodistribution and tumor targeting. The nanorods with medium lengths (<500 nm) had a much longer blood circulation and faster cellular uptake than the nanospheres or long nanorods. Thus, polymeric nanorods with proper lengths may be ideal nanocarriers capable of uniting the opposite requirements in cancer-drug delivery., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A tumor targeting nanoparticle system has been successfully developed to response to the lowered tumor extracellular pH (pHe) and upregulated matrix metalloproteinase 2 (MMP2) in the tumor microenvironment. The nanoparticles are modified with activatable cell-penetrating peptide (designated as dtACPP) that's dual-triggered by the lowered pHe and MMP2. In dtACPP, the internalization function of cell-penetrating peptide (CPP) is quenched by a pH-sensitive masking peptide, linking by a MMP2 substrate. The masking peptide is negatively charged to quench the cationic CPP well after systemic administration. Hence, dtACPP-modified nanoparticles possesses passive tumor targetability via the enhanced permeability and retention (EPR) effect. Once reaching the tumor microenvironment, the pre-existing attraction would be eliminated due to the lowered pHe, accompanying the linker cleaved by MMP2, dtACPP would be activated to expose CPP to drive the nanoparticles' internalization into the intratumoral cells. The studies of plasmid DNA loading, toxicity assessment, cellular uptake, tumor targeting delivery, and gene transfection demonstrate that dtACPP-modified nanoparticle system is a potential candidate for tumor targeting gene delivery., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In order to improve the water-solubility and long-term antibacterial activity of copper nanoparticles (CuNPs), a poly-L-lysine-modified reduced graphene oxide (PLL-rGO) was used as the carrier of CuNPs, and a poly-L-lysine/reduced graphene oxide/copper nanoparticles (PLL-rGO-CuNPs) hybrid was prepared by anchoring the CuNPs on the reduced graphene oxide surface. The novel PLL-rGO-CuNPs hybrid was characterized and the antibacterial activity of it on gram-negative Escherichia coli and Gram-positive Staphylococcus aureus was tested. Such a hybrid showed additively antibacterial activity, and the CuNPs on PLL-rGO were more stable than those on polyvinyl pyrrolidone, resulting in long-term additively antibacterial effect. Meanwhile, this hybrid showed excellent water-solubility, suggesting great potential application in microbial control., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Toll-like receptors (TLRs) play a key role in the recognition of pathogen-associated molecular patterns, including immunostimulatory nucleic acids (INAs). INAs are recognized by TLRs in endosomes, leading to the activation of signalling pathways that activate the innate immune response. This feature makes INAs and their synthetic analogues useful as adjuvants in vaccines and in cancer treatment. We tested a delivery system for the improvement of the therapeutic effect of INAs which consists of a conjugate between transferrin (Tf) and poly-L-lysine (PLL). Tf is a ligand of the transferrin receptor (TfR) and is internalized via receptor-mediated endocytosis, while PLL binds negatively charged INAs. The TfPLL conjugate protected TLR3 ligand polyinosinic:polycytidylic acid [poly(I:C)] from RNase degradation and enhanced the uptake of poly(I:C) in HeLa cells. Co-localization between TfPLL-bound poly(I:C) and lysosomes demonstrated delivery into the endosomal pathway. Time dependence of the production of IL-6 in the primary cell line showed that TfPLL conjugate enabled a gradual release of poly(I:C) and stronger activation of TLR3 receptor in comparison with poly(I:C) alone. Only 3 h of stimulation by poly(I:C) + TfPLL complexes initiated a strong immune response in contrast to poly(I:C) alone. The poly(I:C) + TfPLL complexes have potential use for development of advanced vaccine adjuvants and targeted cancer immune therapy in cells that express higher levels of TfR.

We report that poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymers that bear multiple thiol groups on the polymer backbone are exceptional ligands for gold nanoparticles (AuNPs). In general, these graft copolymer ligands stabilize AuNPs against environments that would ordinarily lead to particle aggregation. To characterize the effect of copolymer structure on AuNP stability, we synthesized thiolated PLL-g-PEGs (PLL-g-[PEG:SH]) with different backbone lengths, PEG grafting densities, and number of thiols per polymer chain. AuNPs were then combined with these polymer ligands, and the stabilities of the resulting AuNP@PLL-g-[PEG:SH] particles against high temperature, oxidants, and competing thiol ligands were characterized using dynamic light scattering, visible absorption spectroscopy, and fluorescence spectrophotometry. Our observations indicate that thiolated PLL-g-PEG ligands combine thermodynamic stabilization via multiple Au-S bonds and steric stabilization by PEG grafts, and the best graft copolymer ligands balance these two effects. We hope that this new ligand system enables AuNPs to be applied to biotechnological applications that require harsh experimental conditions.

Lysine-based polycations are widely used as nonviral carriers for gene delivery. This manuscript reports the results of a comparative study on the in vitro cytotoxicity of a library of three structural polylysine variants, namely, linear polylysine (LPL), dendritic polylysine (DPL), and hyperbranched polylysine (HBPL). The aim of this study was to identify possible effects of polymer molecular weight and architecture on both immediate and delayed cytotoxicity and also to provide a mechanistic understanding for possible differences. Acute cytotoxicities were evaluated using cell viability assays with CHO DG44 cells. At comparable molecular weights, the EC(50) values for the LPL analogues were ∼5-250 times higher as compared to the DPL and HBPL samples. For low molecular weight polycations, osmotic shock was found to be an important contributor to immediate cell death, whereas for the higher molecular weight analogues, direct cell membrane disruption was identified to play a role. Delayed cytotoxicity (≥3 h) was assessed by identifying several of the hallmark events that characterize apoptosis, including phosphatidyl serine translocation, mitochondrial membrane depolarization, cytoplasmic cytochrome C release, and caspase 3 activation. At comparable molecular weights, apoptosis was found to be more pronounced for DPL and HBPL as compared to LPL. This difference was ascribed to the fact that LPL is completely enzymatically degradable, in contrast to DPL and HBPL, which also contain ε-peptidic bonds and are only partially degradable. Because their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA.

A new third generation amphiphilic glycodendrimer was synthesized from a stearylamide lysine dendrimer by condensation of the oligosaccharide moiety. By stepwise condensation and deprotection of di-boc lysine from a core of stearyl amide lysine, a third-generation stearylamide lysine dendrimer was constructed. Acetyl cellobiose and glucose units with the carboxylic acid at the end of alkyl chain attached to the reducing end of the sugar moiety was condensed with surface amino groups of the third generation lysine dendrimer, respectively, to give a new stearylamide acetylcellobiose and acetylglucose lysine dendrimers. The structural analysis was carried out using NMR, IR, and matrix-associated laser desorption/ionization time-of-flight (MALDI TOF) mass spectroscopies. After deacetylation to recover hydroxyl groups and subsequent sulfation, the third-generation sulfated cellobiose stearylamide lysine dendrimer was preliminarily found to have high anti-HIV activity at a 50% effective concentration (EC(50)) as low as 6.4 μg/ml and low cytotoxicity at a 50% cytotoxic concentration (CC(50)) as high as 1000 μg/ml, indicating that the dendrimer gave the enhancement of the functionality of oligosaccharides with low molecular weights. The glycodendrimer with a hydrophobic stearyl chain is immobilized on hydrophobic surfaces by hydrophobic interaction and is expected to provide a new biomedical material with the surface functionality of hydrophilic sulfated oligosaccharides., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A amphiphilic block copolymer composed of conventional monomethoxy (polyethylene glycol)-poly (d,l-lactide-co-glycolide)-poly (l-lysine) (mPEG-PLGA-b-PLL) was synthesized. The chemical structure of this copolymer and its precursors was confirmed by Fourier Transform Infrared Spectroscopy (FTIR), (1)H Nuclear Magnetic Resonance ((1)H NMR) and Gel Permeation Chromatography (GPC). The copolymer was used to prepare nanoparticles (NPs) that were then loaded with either the anti-cancer drug adriamycin or small interfering RNA-negative (siRNA) using a double emulsion method. MTT assays used to study the in vitro cytotoxicity of mPEG-PLGA-b-PLL NPs showed that these particles were not toxic in huh-7 hepatic carcinoma cells. Confocal laser scanning microscopy (CLSM) and flow cytometer analysis results demonstrated efficient mPEG-PLGA-b-PLL NPs-mediated delivery of both adriamycin and siRNA into the cells. In vivo the targeting delivery of adriamycin or siRNA mediated by mPEG-PLGA-b-PLL NPs in the huh-7 hepatic carcinoma-bearing mice was evaluated using a fluorescence imaging system. The targeting delivery results and froze section analysis confirmed that drug or siRNA is deliver to tumor more efficiently by mPEG-PLGA-b-PLL NPs than free drug or Lipofectamine™2000. The high efficiency delivery of mPEG-PLGA-b-PLL NPs mainly due to the enhancement of cellular uptake. These results imply that mPEG-PLGA-b-PLL NPs have a great potential to be used as an effective carriers for adriamycin or siRNA., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

The biodegradable cationic copolymers monomethoxy poly(ethylene glycol)-block-poly(ε-caprolactone)-block-poly(L-lysine) (mPEG-b-PCL-b-PLL, further abbr. as "M") with a PCL block of different lengths and with a triazole linkage between the PCL and PLL blocks were synthesized via ring-opening polymerization of ε-caprolactone and L-lysine N-carboxyanhydride and click reactions. With the copolymer without the PCL block (mPEG-b-PLL, further abbr. as "P") as a control, self-assembling and complexation of these copolymers with siRNA were studied. siRNA-loading capacity, siRNA delivery performance and cellular uptake of the complexation products, namely complex from P (P-complex) and complexes from M1 and M2 (M1-complex and M2-complex), were further examined and correlated to their block-copolymer compositions. The results showed the block copolymers P, M1 and M2 have strong enough binding ability so that the complexes formed can resist the heparin displacement and RNase degradation. Compared to P-complex, M-complexes have smaller size and higher particle density so that they can be internalized via endocytosis more easily than free siRNA or P-complex and they display higher siRNA delivery efficiency and higher gene silencing efficiency than P-complex. The silencing efficiency of micellar complex is close to that of Lipofectamine(TM) 2000 and much better than PEI-25kDa. Therefore, mPEG-b-PCL-b-PLL is expected to be a promising siRNA carrier., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A new type of molecular bottlebrush with poly-L-lysine (PLL) as backbone was synthesized via ROP followed by ATRP. A Nϵ-bromoisobutyryl functionalized Nα-CBZ-L-lysine was firstly synthesized and converted in polymerizable α-amino acid N-carboxyanhydride (NCA), which was then polymerized using Ni(0) transition metal complex to give well-defined bromo-functionalized homopolypeptide (PBrLL), from which we prepared two types of polypeptide bottlebrushes with polystyrene and poly(oligoethylene glycol methacrylate) as side-chains. PBrLL macroinitiator was demonstrated to have high initiation efficiency for ATRP, which allowed good control over side-chain length. CD and FTIR characterization revealed that both PBrLL macroinitiator and PLL backbone of bottlebrushes adopted α-helical conformation in appropriate solvents., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two thermo- and pH-sensitive polypeptide-based copolymers, poly(N-isopropylacrylamide-co-N-hydroxymethylacrylamide)-b-poly(L-lysine) (P(NIPAAm-co-HMAAm)-b-PLL, P1) and poly(N-isopropylacrylamide-co-N-hydroxymethylacrylamide)-b-poly(glutamic acid) (P(NIPAAm-co-HMAAm)-b-PGA, P2), have been designed and synthesized by the ring-opening anionic polymerization of N-carboxyanhydrides (NCA) with amino-terminated P(NIPAAm-co-HMAAm). It was found that the block copolymers exhibit good biocompatibility and low toxicity. As a result of electrostatic interactions between the positively charged PLL and negatively charged PGA, P1 and P2 formed polyion complex (PIC) micelles consisting of polyelectrolyte complex cores and P(NIPAAm-co-HMAAm) shells in aqueous solution. The thermo- and pH-sensitivity of the PIC micelles were studied by UV/Vis spectrophotometry, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Moreover, fluorescent PIC micelles were achieved by introducing two fluorescent molecules with different colors. Photographs and confocal laser scanning microscopy (CLSM) showed that the fluorescence-labeled PIC micelles exhibit thermo- and pH-dependent fluorescence, which may find wide applications in bioimaging in complicated microenvironments., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present a novel photopolymerizable poly(L-lysine) (PLL) and use it to modify polyethylene glycol diacrylate (PEGDA) hydrogels for creating a better, permissive nerve cell niche. Compared with their neutral counterparts, these PLL-grafted hydrogels greatly enhance pheochromocytoma (PC12) cell survival in encapsulation, proliferation, and neurite growth and also promote neural progenitor cell proliferation and differentiation capacity, represented by percentages of both differentiated neurons and astrocytes. The role of efficiently controlled substrate stiffness in regulating nerve cell behavior is also investigated and a polymerizable cationic small molecule, [2-(methacryloyloxy)ethyl]-trimethylammonium chloride (MTAC), is used to compare with this newly developed PLL. The results indicate that these PLL-grafted hydrogels are promising biomaterials for nerve repair and regeneration.

Using solid-phase synthesis, lysine can be oligomerized by a reaction of the peptide carboxylate with the ε-amino group to produce nontoxic, biodegradable cationic peptides, ε-oligo(L-lysines). Here α-substituted derivatives of such ε-oligo(L-lysines) containing arginine and histidine in the side chain were tested as vectors for in vitro gene delivery. Combination of ε-oligolysines with the cationic lipid DOTAP and plasmid DNA resulted in transfection efficiency exceeding that of DOTAP alone, without significant increase in cytotoxicity. Synchrotron small-angle X-ray scattering studies revealed self-assembly of the DOTAP, ε-oligolysines, and DNA to ordered lamellar complexes. High transfection efficiency of the nanoparticles correlates with increase in zeta potential above +20 mV and requires particle size to be below 500 nm. The synergistic effect of branched ε-oligolysines and DOTAP in gene delivery can be explained by the increase in surface charge and by the supramolecular structure of the DOTAP/ε-oligolysine/DNA nanoparticles.

Man-made artificial organic polymers are among the more recent sources of materials used by humans. In medicine, they contribute to applications in surgery, dentistry and pharmacology. Nowadays, innovations in the field of therapeutic polymers rely on novel polymers for specific applications such as guided tissue regeneration, tissue engineering, drug delivery systems, gene transfection, etc. Introducing reactive chemical functions within or along polymer backbones is an attractive route to generate functional polymers for medicine. However, any candidate to effective application must fulfil a number of requirements, grouped under the terms biocompatibility and biofunctionality, to be of real interest and have a future for effective application. Whenever the application requires a therapeutic aid for a limited period of time to help natural healing, bioresorbability is to be taken into account on top of biocompatibility and biofunctionality. This contribution presents the case of "artificial biopolymers" and discusses the potential of some members of the family with respect to temporary therapeutic applications that require functional polymers., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polycations are one of the most frequently used classes of materials for non-viral gene transfer in vivo. Several studies have demonstrated a sensitive relationship between polymer structure and delivery activity. In this work, we used reverse addition-fragmentation chain transfer (RAFT) polymerization to build a panel of N-(2-hydroxypropyl)methacrylamide (HPMA)-oligolysine copolymers with varying peptide length and polymer molecular weight. The panel was screened for optimal DNA-binding, colloidal stability in salt, high transfection efficiency, and low cytotoxicity. Increasing polyplex stability in PBS correlated with increasing polymer molecular weight and decreasing peptide length. Copolymers containing K(5) and K(10) oligocations transfected cultured cells with significantly higher efficiencies than copolymers of K(15). Four HPMA-oligolysine copolymers were identified that met the desired criteria. Polyplexes formed with these copolymers demonstrated both salt stability and transfection efficiencies on-par with poly(ethylenimine) PEI in cultured cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)