Synthesis, characterization and transfection of a novel folate-targeted multipolymeric nanoparticles for gene delivery

Novel folate-conjugated biodegradable multipolymeric nanoparticles (NPs) were constructed and evaluated for potential use in gene delivery to human cervical carcinomas Hela cells, which overexpressed folate receptors. Folate-poly(ethylene glycol)-poly(d, l-lactic-co-glycolic acid) (PELGA-F) was synthesized and collaborated with poly-l-lysine (PLL) to form polymer-polycationic peptide-DNA (PPD) NPs. Fluorescein sodium and polylysine-condensed DNA (PD) were encapsulated in both PELGA nanoparticles (PELGA-NPs) and folate modified nanoparticles (PELGA-F-NPs), which were prepared by a modified solvent extraction/evaporation method. Effects of the folate conjugation and PLL introduction on the uptake of NPs was qualified by fluorescent invert microscopy and quantified by spectrofluorometric measurement, while effect on the gene expression was measured by X-gal staining and luciferase assay, both using Hela cells as an in vitro model. Results showed that cellular uptake of NPs was enhanced by folate modification, but had no difference after PLL encapsulation. In transfection tests, increased gene expression also confirmed the different functions of folate and PLL introduction. It is feasible that folate-linked multipolymeric NPs should be an efficient targeted carrier for gene delivery.     

Preparation of EGFR monoclonal antibody conjugated nanoparticles and targeting to hepatocellular carcinoma

This study aims to determine the sensitivity, specificity and accuracy of epidermal growth factor receptor monoclonal antibody (EGFRmAb) modified poly(lactic acid-co-l-lysine) nanoparticles (PLA-PLL-EGFRmAb) NPs delivery system to EGFR positive cancer cells. In the study, a new PLA-PLL-EGFRmAb NPs was prepared. The cellular cytotoxicity, cellular uptake, and the targeted effect for hepatocellular carcinoma of PLA-PLL-EGFRmAb NPs were investigated. In vitro, the findings of Flow cytometry and Confocal Laser scanning Biological Microscopy showed that PLA-PLL-EGFRmAb NPs can bind to hepatocellular carcinoma cells and were uptaken effectively. In vivo in the SMMC-7721 xenograft mouse model, PLA-PLL-EGFRmAb NPs could target to the tumor effectively, which demonstrated a better targeting. These results showed that the PLA-PLL-EGFRmAb NPs have the potential to be used as a target delivery carrier for tumor therapies.     

Structure and superparamagnetic behaviour of magnetite nanoparticles in cellulose beads

Superparamagnetic magnetite nanoparticles were obtained starting from a mixture of iron(II) and iron(III) solutions in a preset total iron concentration from 0.04 to 0.8 mol l⁻¹ with ammonia at 25 and 70 °C. The regeneration of cellulose from viscose produces micrometrical spherical cellulose beads in which synthetic magnetite were embedded. The characterization of cellulose-magnetite beads by X-ray diffraction, Scanning and Transmission Electron Microscopy and magnetic measurement is reported. X-ray diffraction patterns indicate that the higher is the total iron concentration and temperature the higher is the crystal size of the magnetite obtained. Transmission Electron Microscopy studies of cellulose-magnetite beads revealed the distribution of magnetite nanoparticles inside pores of hundred nanometers. Magnetite as well as the cellulose-magnetite composites exhibit superparamagnetic characteristics. Field cooling and zero field cooling magnetic susceptibility measurements confirm the superparamagnetic behaviour and the blocking temperature for the magnetite with a mean size of 12.5 nm, which is 200 K.     

Preparation and characterization of <Emphasis Type="SmallCaps">l-</Emphasis>cystine and <Emphasis Type="SmallCaps">l-</Emphasis>cysteine intercalated layered double hydroxides

l-cystine and l-cysteine have been intercalated into magnesium–aluminum layered double hydroxide by the methods of coprecipitation and ion-exchange. The structure and composition of the intercalated materials have been characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and elemental analysis. For l-cysteine intercalated composites, two kinds of well-crystallized materials with different basal spacing were obtained, as a result of the different charge on an ion and orientation of the gallery anions. The schematic models of the intercalation structures were proposed. In addition, the thermal decomposition of l-cystine and l-cysteine intercalated LDHs has been investigated by means of thermogravimetry and differential thermal analysis (TG-DTA).     

Facile preparation of magnetic sodium alginate/carboxymethyl cellulose composite hydrogel for removal of heavy metal ions from aqueous solution

A novel magnetic polysaccharide composite hydrogel was successfully constructed by using sodium alginate (SA) and carboxymethyl cellulose (CMC) as the backbone and filled with in situ Fe₃O₄ nanoparticles, which was then employed for removal of heavy metal ion from aqueous solution. The obtained magnetic SA/CMC composite hydrogel was characterized by Fourier transform infrared spectroscopy, fluorescence microscope, thermogravimetric and vibrating sample magnetometer. Effect of contact time, pH and adsorbent dosage on the adsorption of heavy metal ions by the magnetic SA/CMC hydrogel have also been studied. The results show that the prepared magnetic SA/CMC hydrogel can be effectively utilized in the removal of heavy metal ions from aqueous solution. The maximal adsorption capacity of Mn(II), Pb(II), and Cu(II) as calculated from the Langmuir model were 71.83, 89.49, and 105.93 mg/g, respectively. The adsorption process of the magnetic SA/CMC hydrogel on the heavy metal ions can be attributed to ion exchange and chemical adsorption. What’s more, the magnetic hydrogel exhibited high efficiency after four cycles, which indicating it offers great potential for practical application in the removal of heavy metal ions from aqueous solution.

     

Surface tailoring of poly(ethylene terephthalate) via ligand-tethered comb-like PEG to enhance endothelialization

The comb-like PEG (CPEG) end-tethered with l-lysine was explored to surface modification of PET to enhance endothelialization. The hydroxyl end groups of CPEG were oxygenated into aldehyde groups. The CPEG-CHO was grafted onto the aminolysized PET. The l-lysine was then end-tethered onto surface via the residual aldehyde groups. The surface modification was confirmed by ATR-FTIR, contact angle and XPS measurements. The endothelial cell adhesion, proliferation and viability results indicated that the PET-CPEG resisted cell adhesion and growth, where as PET-CPEG-lysine promoted cell adhesion and growth. The MTT assay and total cell protein tests indicated that the endothelial cells on PET-CPEG-lysine had high viability. Cell spread uniformly and covered completely on the PET-CPEG-lysine. The CPEG end tethered with l-lysine could regulate cell adhesion and growth and enhance surface endotheliazation.     

A Nanoparticle Ink Allowing the High Precision Visualization of Tissue Engineered Scaffolds by MRI (Small 30/2023)

Hydrogels are widely used as cell scaffolds in several biomedical applications. Once implanted in vivo, cell scaffolds must often be visualized, and monitored overtime. However, cell scaffolds appear poorly contrasted in most biomedical imaging modalities such as magnetic resonance imaging (MRI). MRI is the imaging technique of choice for high-resolution visualization of low-density, water-rich tissues. Attempts to enhance hydrogel contrast in MRI are performed with “negative” contrast agents that produce several image artifacts impeding the delineation of the implant's contours. In this study, a magnetic ink based on ultra-small iron oxide nanoparticles (USPIONs; <5 nm diameter cores) is developed and integrated into biocompatible alginate hydrogel used in cell scaffolding applications. Relaxometric properties of the magnetic hydrogel are measured, as well as biocompatibility and MR-visibility (T₁-weighted mode; in vitro and in vivo). A 2-week MR follow-up study is performed in the mouse model, demonstrating no image artifacts, and the retention of “positive” contrast overtime, which allows very precise delineation of tissue grafts with MRI. Finally, a 3D-contouring procedure developed to facilitate graft delineation and geometrical conformity assessment is applied on an inverted template alginate pore network. This proof-of-concept establishes the possibility to reveal precisely engineered hydrogel structures using this USPIONs ink high-visibility approach.     

Template and template-free preparation of one-dimensional metallic nanostructures

In this article, we have studied and developed two approaches for organizing metallic nanoparticles into one-dimensional assemblies. The first uses DNA as a ‘template’ and allows the preparation of various silver nanostructures (‘beads-on-a-string’ or rod-like wires). The conductance of such nanostructures was demonstrated by employing a powerful technique, Electrostatic Force Microscopy (EFM). This technique gave us ‘contactless’ information about the electrical properties of silver nanostructures, aligned on a SiO₂/Si surface. Additionally, I–V characteristics of a single silver nanowire crossing two microelectrodes were recorded. The nanowire resistivity was estimated at 1.46 × 10⁻⁷ Ω m (at 300 K), which is one order of magnitude higher than that of bulk silver (1.6 × 10⁻⁸ Ω m). The second approach is a ‘template-free’ one, and exploits the binding ability of l-arginine, which favours the self-assembling of capped gold nanoparticles into gold nanochains. The results suggest that gold nanochains were formed due to dipole–dipole interaction between adjacent nanoparticles, which fuse together through an oriented attachment mechanism. Atomic force microscopy, TEM, UV–vis spectroscopy and X-ray diffraction were used to characterize the morphological, optical and structural properties of these metallic nanostructures.     

Computational and electrochemical studies of some amino acid compounds as corrosion inhibitors for mild steel in hydrochloric acid solution

The corrosion inhibition behaviour of four selected amino acid compounds, namely l-cysteine, l-histidine, l-tryptophan and l-serine on mild steel surface in deaerated 1 M HCl solution were studied electrochemically by Tafel polarization and electrochemical impedance spectroscopy methods and computationally by the quantum chemical calculation and molecular dynamics simulation. Electrochemical results show that these amino acid compounds inhibit the corrosion of mild steel in 1 M HCl solution significantly. The order of inhibition efficiency of these inhibitors follows the sequence: l-tryptophan > l-histidine > l-cysteine > l-serine. The quantum chemical calculations were performed to characterize the electronic parameters which are associated with inhibition efficiency. The molecular dynamics simulations were applied to find the equilibrium adsorption configurations and calculate the interaction energy between inhibitors and iron surface. Results obtained from Tafel and impedance methods are in good agreement. The electrochemical experimental results are supported by the theoretical data.     

Study of impregnation of poly(l-lactide-ran-ε-caprolactone) copolymers with useful compounds in supercritical carbon dioxide

Outstanding controlled release materials were developed using statistically random copolymers of l-lactide (l-LA) with ε-caprolactone (CL) using Sn(oct)₂ as a catalyst at 150 °C for 24 h without solvent. Preparation of novel controlled release materials was carried out using useful organic compounds with low boiling points and synthetic random copolymers composed of l-LA and CL as base materials under supercritical carbon dioxide (scCO₂). Low-boiling-point compounds such as d-limonene, hinokitiol, and trans-2-hexenal were used. In impregnation experiments using scCO₂, the amounts of low-boiling-point compounds increased with an increase in l-LA content. When enzymatic degradation of poly(l-LA-ran-CL) was performed using lipase PS, copolymers with higher CL contents degraded more rapidly than did copolymers with higher l-LA content. In contrast, enzymatic degradation of copolymers occurred to a higher degree with increased l-LA content in enzymatic degradation by proteinase K. In a controlled release experiment with poly(l-LA-ran-CL) (=73/27), after 400 h of degradation by proteinase K, the remaining weight of the copolymer pellet was 6% and the amount of d-limonene remaining in the pellet was 15%.     

Autologous urothelial cells transplantation onto a prefabricated capsular stent for tissue engineered ureteral reconstruction

In this study, we have fabricated an artificial ureter by transplantation of in vitro-expanded urothelial cells onto an in vivo-prefabricated capsular stent using tissue engineering methods. Spiral poly (l-lactic acid) (PLLA) stents were transplanted into the subcutaneous of Wistar rats for a period of 1, 2 or 3 weeks to induce the formation of connective tissue capsules on their surfaces. The capsular PLLA stents were then decellularized and further recellularized with bladder epithelial cells to fabricate artificial ureters. The results showed that the entrapped cells in all capsules remained continuously proliferation and lined up in continuous layers. In addition, the urothelial cells on the capsular stents with an embedding period of 2 or 3 weeks showed higher proliferative viability compared with the cells on the stents with an embedding time of 1 week (P < 0.05). The results of the study indicated that the prefabricated capsular stents could serve as alternative cell carriers for tissue engineered ureters, especially with embedding time from 2 to 3 weeks.     

<Emphasis Type="SmallCaps">l</Emphasis>-Tryptophan as green corrosion inhibitor for low carbon steel in hydrochloric acid solution

The inhibition behavior of low carbon steel in 1 M HCl by l-tryptophan was investigated with weight loss experiment and Tafel polarization curve in the used temperature range (298–328 K). All the experimental results show that l-tryptophan has excellent corrosion inhibition performance and the most effective concentration of inhibitor is 1 × 10⁻² mol L⁻¹. The Tafel polarization curve results indicate that l-tryptophan acts more as a cathodic than anodic inhibitor. The adsorption of l-tryptophan on the surface of low carbon steel obeys the Langmuir adsorption isotherm, and the thermodynamic parameters were determined and discussed. The adsorption behavior of l-tryptophan at Fe surface (1 1 0) was also investigated by the molecule dynamics simulation method and density functional theory. The results indicated that the l-tryptophan could adsorb firmly on the Fe surface through the indole ring with π-electrons and nitrogen/oxygen atom with lone-pair electrons in its molecule.     

Supercritical CO<Subscript>2</Subscript> processing of polymers for the production of materials with applications in tissue engineering and drug delivery

Supercritical carbon dioxide (SCCO₂) was used for the preparation of foamed sponges and intermingled fibers of biopolymers with potential applications in tissue engineering and drug delivery. The work was focused on the processing of both biodegradable polylactic acid (l-PLA) and non-biodegradable polymethylmethacrylate (PMMA) homopolymers. Monolithic porous sponges of amorphous PMMA were prepared using SCCO₂ as a porogen agent by simple swelling and foaming. Under similar experimental conditions, l-PLA was crystallized. The study also addresses the impregnation of biopolymers with an active agent dispersed in SCCO₂. The drug used for impregnation was triflusal, a platelet antiaggregant inhibitor for thrombogenic cardiovascular diseases. Foaming often leads to a closed pore structure after depressurization which is disadvantageous for 3D scaffolds as it does not fulfill the requirement of interconnectivity necessary for cell migration. To overcome these drawbacks, fibers forming macroporous structures were prepared using a semicontinuous antisolvent (SAS) technique.     

In vitro biocompatibility testing of polylactides

Four high-molecular weight polylactides, three poly-l-lactides of 100, 250 and 500 kDa and a poly-dl-lactide of 400 kDa, were tested qualitatively in vitro. Cells were cultured on polylactide films and with media based on the artificially aged polylactides, as well as with different concentrations of the final degradation products (monomers). Implant site-related cell types were selected. Three kinds of epithelial cells (middle ear, ear canal and nasal septum) as well as fibroblasts and osteosarcoma cells were used. Characteristic of all four polylactides was the normal morphology of cells when cultured on polylactide films and with the artificially ageing media. Although the polylactide films revealed still uncovered spots after 14 days culture, the control cultures were already confluent. The different concentrations of monomers revealed normal cell morphologies except for the 10 mg ml^-1 concentration, which showed larger fibroblasts, and the nasal septum epithelium showed more signs of terminal differentiation for the 10 mg ml^-1 d-monomer than was seen for the l-monomer even if the osmolarity was adjusted. The degradation products of polylactide are not expected to cause adverse reactions when implanted, since cell cultures with monomer concentrations up to 1 mg ml^-1 resulted in normal morphologies. In the present study the cells were not able to cover the polylactide films completely.     

Tissue biocompatibility of new biodegradable drug-eluting stent materials

Drug-eluting stents are a recent innovation for endovascular and endourethral purposes. The aim of this study was to assess the biocompatibility of new biodegradable drug-eluting stent materials in vivo. Rods made of SR-PLDLA (self-reinforced poly-96l,4d-lactic acid) covered with P(50l/50d)LA and rods made of 96l/4D SR-PLA and covered with P(50l/50d)LA including indomethacin 3.3 μg/mm² or dexamethasone 1.5 μg/mm², were inserted into the dorsal muscles of 20 rabbits serving as test animals. Rods made of silicone and organotin-positive polyvinylchloride were used as negative and positive controls. The animals were sacrificed after 1 week, 1 month, 2 months or 4 months. Histological changes attributable to the operative trauma were seen in all specimens at 1 week and 1 month. At 2 months both dexamethasone and indomethacin induced less fibrosis than the plain SR-PLDLA covered with P(50l/50d)LA without drug. At 4 months dexamethasone induced both chronic inflammatory changes and foreign body reaction, whereas the reactions in the indomethacin and drug-free plain SR-PLDLA groups were insignificant. The new biodegradable drug-eluting stent materials are highly biocompatible. Drug-eluting biodegradable stents may offer a promising new treatment modality for vascular and urethral diseases. However, further studies are needed to demonstrate their feasibility and efficacy.     

Morphology and rheology of poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactide)/polystyrene blends filled with silica nanoparticles

The successful development of co-continuous structure from poly(l-lactide) (PLLA) blends by melt mixing with lower PLLA content is highly desired in preparing macroporous biomaterials. However, the low viscosity of PLLA makes it difficult to prepare co-continuous PLLA blends at low PLLA concentration. In this study, hydrophilic silica nanoparticle is adopted to control the morphology of co-continuous polystyrene (PS)/PLLA blends. The influence of nanoparticle concentration on the co-continuity intervals and rheological properties of PS/PLLA blends are examined. The morphological stability of blends against melt annealing is also determined and discussed with a conceptual coarsening model for co-continuous structure. The results demonstrate that the incorporation of silica nanoparticles into PS/PLLA blends can be used to prepare macroporous PLLA structure with controllable pore size at lower PLLA content.     

Synthesis and characterization of arginine–glycine–aspartic peptides conjugated poly(lactic acid-co-<Emphasis Type="SmallCaps">l</Emphasis>-lysine) diblock copolymer

A biodegradable Copolymer of poly(lactic acid-co-lysine)(PLA–PLL) was synthesized by a modified method and novel Arginine–Glycine–Aspartic (RGD) peptides were chemical conjugated to the primary ε-amine groups of lysine components in four steps: I to prepare the monomer of 3-(N^ε-benzoxycarbonyl-l-lysine)-6-l-methyl-2,5-morpholinedione; II to prepare diblock copolymer poly(lactic acid-co-(Z)-l-lysine) (PLA–PLL(Z)) by ring-opening polymerization of monomer and l,l-lactide with stannous octoate as initiator; III to prepare diblock copolymer PLA–PLL by deprotected the copolymer PLA–PLL(Z) in HBr/HoAc solution; IV the reaction between RGD and the primary ε-amine groups of the PLA–PLL. The structure of PLA–PLL–RGD and its precursors were conformed by FTIR-Raman and ¹H NMR. Low weight average molecular weight (9,200 g/mol) of the PLA–PLL was obtained and its PDI is 1.33 determined by GPC. The PLA–PLL contained 2.1 mol% lysine groups as determined by ¹H NMR using the lysine protecting group’s phenyl protons. Therefore, the novel RGD-grafted diblock copolymer is expected to find application in drug carriers for tumor therapy or non-viral DNA carriers for gene therapy.     

Carbonization behavior of <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan and gluten

Carbonization behavior of l-tryptophan and gluten has been investigated in comparison with that of acenaphthylene using CHN elemental analysis, wide angle X-ray diffractometry, laser Raman spectroscopy and polarizing light microscopy. The carbon derived from l-tryptophan by the heat-treatment at 3,000 °C showed almost the same degree of graphitization as that from acenaphthylene and the average interlayer spacings of both these carbons approached to 0.3354 nm. The average interlayer spacing of the carbon from gluten, on the other hand, did not approach to this value at 3,000 °C. The crystallites in the carbon from l-tryptophan were smaller than those in the carbon from acenaphthylene but larger than those in the carbon from gluten. The ratio, R, of the intensity of the Raman band at 1,360 cm⁻¹ against that at 1,580 cm⁻¹ and the half width, Δλ, of the Raman band at 1,580 cm⁻¹ were measured. The R and Δλ are the measures for the degree of graphitization. Those values for the carbon from l-tryptophan were nearly equal to those for the carbon from acenaphthylene and smaller than those for the carbon from gluten. The thin film of l-tryptophan heat-treated at 500 °C for 2 h showed a texture consisting of a fine mosaic mesophase structure and an anisotropic flow-type texture of mesophase.     

Titanium dioxide (TiO<Subscript>2</Subscript>) nanoparticles filled poly(<Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis> lactid acid) (PDLLA) matrix composites for bone tissue engineering

Titanium dioxide (TiO₂) nanoparticles were investigated for bone tissue engineering applications with regard to bioactivity and particle cytotoxicity. Composite films on the basis of poly(d,l lactid acid) (PDLLA) filled with 0, 5 and 30 wt% TiO₂ nanoparticles were processed by solvent casting. Bioactivity, characterised by formation of hydroxyapatite (HA) on the materials surface, was investigated for both the free TiO₂ nanoparticles and PDLLA/TiO₂ composite films upon immersion in supersaturated simulated body fluid (1.5 SBF) for up to 3 weeks. Non-stoichiometric HA nanocrystals (ns-HA) with an average diameter of 40 nm were formed on the high content (30 wt% TiO₂) composite films after 2 weeks of immersion in 1.5 SBF. For the pure PDLLA film and the low content composite films (5 wt% TiO₂) trace amounts of ns-HA nanocrystals were apparent after 3 weeks. The TiO₂ nanopowder alone showed no bioactivity. The effect of TiO₂ nanoparticles (0.5–10,000 μg/mL) on MG-63 osteoblast-like cell metabolic activity was assessed by the MTT assay. TiO₂ particle concentrations of up to 100 μg/mL had no significant effect on MG-63 cell viability.     

Enzymatic degradation of the hydrogels based on synthetic poly(α-amino acid)s

Biodegradable hydrogels are studied as potential scaffolds for soft tissue regeneration. In this work biodegradable hydrogels were prepared from synthetic poly(α-amino acid)s, poly(AA)s. The covalently crosslinked gels were formed by radical copolymerization of methacryloylated poly(AA)s, e.g. poly[N ⁵-(2-hydroxy-ethyl)-l-glutamine-ran-l-alanine-ran-N ⁶-methacryloyl-l-lysine], as a multifunctional macro-monomer with a low-molecular-weight methacrylic monofunctional monomer, e.g. 2-hydroxyethyl methacrylate (HEMA). Methacryloylated copolypeptides were synthesized by polymerization of N-carboxyanhydrides of respective amino acids and subsequent side-chain modification. Due to their polypeptide backbone, synthetic poly(AA)s are cleavable in biological environment by enzyme-catalyzed hydrolysis. The feasibility of enzymatic degradation of poly(AA)s alone and the hydrogels made from them was studied using elastase, a matrix proteinase involved in tissue healing processes, as a model enzyme. Specificity of elastase for cleavage of polypeptide chains behind the l-alanine residues was reflected in faster degradation of l-alanine-containing copolymers as well as of hydrogels composed of them.