Incorporation of Indole Significantly Improves the Transfection Efficiency of Guanidinium-Containing Poly(Methacrylamide)s.

A highly efficient transfection agent is reported that is based on terpolymer consisting of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl) methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by analogy to the amphipathic cell-penetrating peptides containing tryptophan and arginine residues. The incorporation of the indole-bearing monomer leads to successful plasmid DNA condensation even at a nitrogen-to-phosphate (N/P) ratio of 1. The hydrodynamic diameter of polyplexes is determined to be below 200 nm for all N/P ratios. The transfection studies demonstrate a 200-fold increase of the transgene expression in comparison to P(HPMA-co-GPMA) with the same guanidinium content. This study reveals the strong potential of the indole group as a side-chain pendant group that can increase the cellular uptake of polymers and the transfection efficiency of the respective polyplexes.

Tackling the Limitations of Copolymeric Small Interfering RNA Delivery Agents by a Combined Experimental-Computational Approach.

Despite the first successful applications of nonviral delivery vectors for small interfering RNA in the treatment of illnesses, such as the respiratory syncytial virus infection, the preparation of a clinically suitable, safe, and efficient delivery system still remains a challenge. In this study, we tackle the drawbacks of the existing systems by a combined experimental-computational in-depth investigation of the influence of the polymer architecture over the binding and transfection efficiency. For that purpose, a library of diblock copolymers with a molar mass of 30 kDa and a narrow dispersity (D < 1.12) was synthesized. We studied in detail the impact of an altered block size and/or composition of cationic diblock copolymers on the viability of each respective structure as a delivery agent for polynucleotides. The experimental investigation was further complemented by a computational study employing molecular simulations as well as an analytical description of systemic properties. This is the first report in which molecular dynamics simulations of RNA/cationic polymer complexes have been performed. Specifically, we developed and employed a coarse-grained model of the system at the molecular level to study the interactions between polymer chains and small interfering RNA. We were further able to confirm a threshold lengthbinding block/lengthnonbinding block ratio, which is required for efficient complexation of siRNA, and it was possible to find a correlation between the length of the cationic block and the size of the resulting polyplex. Hence, the combined insights from the experiments and the theoretical investigation resulted in a wealth of information about the properties of cationic diblock copolymers employed as RNA delivery agents, in particular regarding the molecular and mechanistic details of the interaction between the two components of a polyplex.

New Techniques to Assess In Vitro Release of siRNA from Nanoscale Polyplexes.

PURPOSE: Release of siRNA from nanoscale polyplexes is a crucial yet little investigated process, important during all stages of therapeutic research. Here we develop new methods to characterize polyplex stability early on in the development of new materials. METHODS: We used double fluorescent labeled siRNA to compare binding and stability of a panel of chemically highly diverse nanoscale polyplexes, including peptides, lipids, nanohydrogels, poly-L-lysine brushes, HPMA block copolymers and manganese oxide particles. Conventional EMSA and heparin competition methods were contrasted with a newly developed microscale thermophoresis (MST) assay, a near-equilibrium method that allows free choice of buffer conditions. Integrity of FRET-labeled siRNA was monitored in the presence of nucleases, in cell culture medium and inside living cells. This approach characterizes all relevant steps from polyplex stability, over uptake to in vitro knockdown capability. RESULTS: Diverging polyplex binding properties revealed drawbacks of conventional EMSA and heparin competition assays, where MST and FRET-based siRNA integrity measurements offered a better discrimination of differential binding strength. Since cell culture medium left siRNA in all polyplexes essentially intact, the relevant degradation events could be pinpointed to occur inside cells. Differential binding strength of the variegated polyplexes correlated only partially with intracellular degradation. The most successful compounds in RNAi showed intermediate binding strength in our assays. CONCLUSIONS: We introduce new methods for the efficient and informative characterization of siRNA polyplexes with special attention to stability. Comparing FRET-labeled siRNA in different polyplexes associates successful knockdown with intermediate siRNA stability in various steps from formulation to intracellular persistence.

The guanidinium group as a key part of water-soluble polymer carriers for siRNA complexation and protection against degradation.

Here, the preparation of a novel block copolymer consisting of a statistical copolymer N-(2-hydroxypropyl) methacrylamide-s-N-(3-aminopropyl) methacrylamide and a short terminal 3-guanidinopropyl methacrylamide block is reported. This polymer structure forms neutral but water-soluble nanosized complexes with siRNA. The siRNA block copolymer complexes are first analyzed using agarose gel electrophoresis and their size is determined with fluorescence correlation spectroscopy. The protective properties of the polymer against RNA degradation are investigated by treating the siRNA block copolymer complexes with RNase V1. Heparin competition assays confirm the efficient release of the cargo in vitro. In addition, the utilization of microscale thermophoresis is demonstrated for the determination of the binding strength between a fluorescently labeled polyanion and a polymer molecule.

The influence of gradient and statistical arrangements of guanidinium or primary amine groups in poly(methacrylate) copolymers on their DNA binding affinity.

Herein, we report the first gradient guanidinium containing cationic copolymers and investigate their binding ability to plasmid DNA (pDNA). To understand the effect of different charge distributions and cationic charge sources (primary amines vs. guanidinium group) on (pDNA) binding affinity, we synthesized a library of well-defined statistical cationic copolymers comprising N-(2-hydroxy-propyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA) or N-(3-guanidinopropyl)methacrylamide (GPMA) and compared them with gradient polymers containing the same monomers of similar composition. All copolymers were synthesized through aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization at various monomer ratios by aiming at similar molar masses with low dispersity indices. For the molar mass characterization, in addition to size exclusion chromatography with two different systems, hydrodynamic characterization utilizing analytical ultracentrifugation, viscometry, and accompanied density measurements was conducted. pDNA was used as a model drug to demonstrate the impact of copolymer architecture on binding efficiency. For both HPMA-APMA and HPMA-GPMA copolymers, the gradient distribution demonstrated superior binding and denser packing of pDNA than their statistical counterparts at 20% and lower cationic charge contents. With respect to charge origin, the guanidinium group represented a higher binding efficiency than primary amines with the same nitrogen to phosphate ratio (N/P ratio). Our study demonstrates the profound effect of gradient monomer arrangement on the ability of polyplex formation and reveals the potential for further investigation in gene delivery applications. Gradient guanidinium containing copolymers have great promise for gene delivery applications due to their high affinity toward pDNA even at very low degrees (<20%) of charged monomer content.

Overcoming the barrier of CD8+T cells: Two types of nano-sized carriers for siRNA transport.

Bioengineering immune cells via gene therapy offers treatment opportunities for currently fatal viral infections. Also cell therapeutics offer most recently a breakthrough technology to combat cancer. These primary human cells, however, are sensitive to toxic influences, which make the utilization of optimized physical transfection techniques necessary. The otherwise commonly applied delivery agents such as LipofectamineⓇ or strongly cationic polymer structures are not only unsuitable for in vivo experiments, but are also highly toxic to immune cells. This study aimed to improve the design of polymeric carrier systems for small interfering RNA, which would allow efficient internalization into CD8+T-cells without affecting their viability and thereby removing the current limitations in the field. Here, two new carrier systems for small interfering RNA were tested. One is a cationic diblock copolymer, in which less than 10% of the monomers were modified with triphenylphosphonium cations. This moiety is lipophilic, promotes uptake and it is mostly known for its mitotropic properties. Furthermore, cationic nanohydrogel particles were synthesized in exceedingly small sizes (Rh < 14 nm). After full physicochemical characterization of the two carriers, extensive cytotoxicity studies were performed and the concentration dependent uptake into CD8+T-cells was tested in correlation to incubation time and protein content of the surrounding medium. Both carriers facilitated efficient complexation of siRNA as well as significant internalization into primary human cells in less than three hours of incubation. In addition, neither of the delivery systems reduced cell viability making them good candidates to transport siRNA into CD8+T-cells efficiently. STATEMENT OF SIGNIFICANCE: This study provides insights into the design of polymeric delivery agents as the method of choice for overcoming the limitations of cell manipulation. Until now, CD8+T-cells, which have become a treatment tool for currently fatal diseases, have not yet been made accessible for gene silencing by polymeric siRNA carrier systems. Choosing appropriate modification approaches for two chemically different polymer structures, we were, in both cases, able to achieve significant uptake in these cells even at low concentrations and without inducing cytotoxicity. These results remove current limitations and pave the way for bioengineering via gene therapy.

Ultra-sensitive detection of malathion using quantum dots-polymer based fluorescence aptasensor.

A novel detection platform with high malathion specificity has been developed, which operates based on the signal response in the fluorescence of CdTe@CdS quantum dots (QDs). The designed nanoprobe comprises of QDs, poly(N-(3-guanidinopropyl)methacrylamide) homopolymer (PGPMA) and malathion specific aptamer. The interaction of aptamer with malathion results in switching off of the fluorescence signal of the probe due to the availability of the cationic polymer, which causes quenching of the QDs. However, in the absence of malathion, the polymer interacts with the aptamer, via electrostatic interactions thereby rendering the fluorescence of QDs unaffected. The assay exhibited excellent sensitivity towards malathion with a detection limit of 4pM. A logarithmic correlation was observed in a wide range of malathion concentrations from 0.01nm to 1µM, facilitating the potential of proposed assay in the quantitative determination of the analyte of interest. The selectivity of the designed probe was confirmed in the presence of various pesticides, commonly employed in agricultural fields.

Overcoming drug resistance by cell-penetrating peptide-mediated delivery of a doxorubicin dimer with high DNA-binding affinity.

We describe the synthesis and characterization of a novel bioconjugate, consisting of an octaarginine cell-penetrating peptide and a highly DNA-affine doxorubicin dimer. The linkage between the two components is composed of a cleavable disulfide bond, which enables the efficient intracellular delivery of the cytotoxic payload within the reductive environment of the cytosol, mediated through glutathione. To determine the DNA-binding affinity of the dimeric drug molecule, microscale thermophoresis was applied. This is the first utilization of this method to assess the binding interactions of an anthracycline drug with nucleic acids. The cytotoxic effect of the peptide-drug conjugate, studied with drug-sensitive and doxorubicin-resistant cancer cells, demonstrates that the bioconjugate can successfully overcome drug resistance in neuroblastoma cells.