Indole, Phenyl, and Phenol Groups: The Role of the Comonomer on Gene Delivery in Guanidinium Containing Methacrylamide Terpolymers.

This report highlights the importance of hydrophobic groups mimicking the side chains of aromatic amino acids, which are tryptophan, phenylalanine, and tyrosine, in guanidinium bearing poly(methacrylamide)s for the design of non-viral gene delivery agents. Guanidinium containing methacrylamide terpolymers are prepared by aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization with different hydrophobic monomers, N-(2-indolethyl)methacrylamide (IEMA), N-phenethylmethacrylamide (PhEMA), or N-(4-hydroxyphenethyl)methacrylamide (PhOHEMA) by aiming similar contents. The well-defined polymers are obtained with a molar mass of ≈15 000 g mol-1 and ≈1.1 dispersity. All terpolymers demonstrate almost comparable in vitro cell viability and hemocompatibility profiles independent of the type of side chain. Although they all form positively charged, enzymatically stable polyplexes with plasmid DNA smaller than 200 nm, the incorporation of the IEMA monomer improve these parameters by demonstrating a higher DNA binding affinity and forming nanoassemblies of about 100 nm. These physicochemical characteristics are correlated with increased transfection rates in CHO-K1 cells dependent on the type of the monomer and the nitrogen to phosphate (N/P) ratio of the polyplexes, as determined by luciferase reporter gene assays.

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.

Chitosan functionalized doxorubicin loaded poly(methacrylamide) based copolymeric nanoparticles for enhanced cellular internalization and in vitro anticancer evaluation.

Doxorubicin (Dox), a chemotherapeutic agent, encounters challenges such as a short half-life, dose-dependent toxicity, and low solubility. In this context, the present study involved the fabrication of N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(3-aminopropyl)methacrylamide (APMA) bearing P(HPMA-s-APMA) copolymeric nanoparticles (P(HPMA-s-APMA) NPs) and their investigation for efficient delivery of Dox. Furthermore, the synthesized nanoparticles (NPs) were coated with chitosan (Cht) to generate positively charged nanoformulations. The prepared formulations were evaluated for particle size, morphology, surface charge analysis, percentage encapsulation efficiency (EE%), and drug release studies. The anticancer activity of Cht-P(HPMA-s-APMA)-Dox NPs was assessed in the HeLa cancer cell line. The prepared P(HPMA-s-APMA)-Dox NPs exhibited an average particle size of 240-250 nm. Chitosan decorated P(HPMA-s-APMA)-Dox NPs displayed a significant increase in particle size, and the zeta potential shifted from negative to positive. The EE% for Cht-P(HPMA-s-APMA)-Dox NPs was calculated to be 68.06 %. The drug release studies revealed a rapid release of drug from Cht-P(HPMA-s-APMA)-Dox NPs at pH 4.8 than pH 7.4, demonstrating the pH-responsiveness of nanoformulation. Furthermore, the cell viability assay and internalization studies revealed that Cht-P(HPMA-s-APMA)-Dox NPs had a high cytotoxic response and significant cellular uptake. Hence, the Cht-P(HPMA-s-APMA)-Dox NPs appeared to be a suitable nanocarrier for effective, and safe chemotherapy.

Ultrasensitive aptasensor for arsenic detection using quantum dots and guanylated Poly(methacrylamide).

The present study demonstrate the first time usage of poly (HPMA-s-GPMA) copolymer for the fabrication of three-component based aptasensor for simple, selective, rapid and label free detection of arsenite (As3+). For this purpose, guanidinium bearing poly (HPMA-s-GPMA) copolymer and MPA-CdTe@CdS quantum dots (QDs) was employed in conjunction with As3+ specific aptamer. This protocol utilizes the quenching phenomena displayed by QDs due to the competitive binding of As3+ ions and cationic copolymer to the aptamer. In particular, the As3+ bind to the specific aptamer, leaving poly (HPMA-s-GPMA) freely available for its electrostatic intercations with QDs, which quenches the fluorescent signal. Contrarily, in the absence of As3+ ions, the aptamer can electrostatically bind to poly (HPMA-s-GPMA); making copolymer inactive to affect the fluorescence signal of the QDs. The efficiency of the proposed fluorescence nanoprobe was further tested using linear calibration curves. The obtained data in the range of 0.01-100 nM showed excellent specificity for As3+ ions with the limit of detection (LOD) of 246.77 pM. Moreover, the "on-off" fluorescent aptasensor is highly selective for As3+ ions in the presence of other interfering metal ions by utilizing As3+ specific aptamer. Furthermore, the reported study showed outstanding applicability in the real-world samples (water, food and soil) containing preservatives, metal ions, minerals, and other moieties. The proposed sensing platform not only exhibits the trace level detection of As3+ ions in cost-effective manner but also opens a pathway for the development of state-of-art device fabrication for on-site detection of arsenic.

PEGylation of Guanidinium and Indole Bearing Poly(methacrylamide)s - Biocompatible Terpolymers for pDNA Delivery.

This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative - poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9 MA)) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9 MA) is varied from 3 to 40 kg mol-1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9 MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9 MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9 MA) up to 10 kg mol-1 . Moreover, a fluorescently labeled analogue of P(MEO9 MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol-1 of P(MEO9 MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).

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.