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 K5 and K10 oligocations transfected cultured cells with significantly higher efficiencies than copolymers of K15. Four HPMA-oligolysine copolymers were identified that met desired criteria. Polyplexes formed with these copolymers demonstrated both salt stability and transfection efficiencies on-par with poly(ethylenimine) PEI in cultured cells.

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Self-amplifying RNA (SAM) represents a versatile tool that can be used to develop potent vaccines, potentially able to elicit strong antigen-specific humoral and cellular-mediated immune responses to virtually any infectious disease. To protect the SAM from degradation and achieve efficient delivery, lipid nanoparticles (LNPs), particularly those based on ionizable amino-lipids, are commonly adopted.Herein, we compared commonly available cationic lipids, which have been broadly used in clinical investigations, as an alternative to ionizable lipids. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG) was used. The cationic lipids investigated including 3ß-[N-(N',N'dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDA), 1,2dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) and N-(4-carboxybenzyl)-N,Ndimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ). Whilst all cationic LNP (cLNP) formulations promoting high association with cells in vitro, those formulations containing the fusogenic lipid 1,2dioleoyl-sn-3-phosphoethanolamine (DOPE) in combination with DOTAP or DDA were the most efficient at inducing antigen expression. Therefore, DOTAP and DDA formulations were selected for further in vivo studies and were compared to benchmark ionizable LNPs (iLNPs). Biodistribution studies revealed that DDA-cLNPs remained longer at the injection site compared with DOTAP-cLNPs and iLNPs when administered intramuscularly in mice. However, both the cLNP formulations and the iLNPs induced strong humoral and cellular-mediated immune responses in mice that were not significantly different at a 1.5 µg SAM dose. In summary, cLNPs based on DOTAP and DDA are an efficient alternative to iLNPs to deliver SAM vaccines.

A new paradigm was designed for apoptosis induction mediated by the biorecognition of coiledcoil motifs at the Raji B cell surface. The heterodimerization of complementary peptides, one bound to Fab' antibody fragment, the other as grafts to HPMA copolymer, results in crosslinking of CD20 target antigens, and consequently, initiation of apoptosis.

Non-viral gene delivery systems capable of transfecting cells in the brain are critical in realizing the potential impact of nucleic acid therapeutics for diseases of the central nervous system. In this study, the membrane-lytic peptide melittin was incorporated into block copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The first block, designed for melittin conjugation, was composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) and the second block, designed for DNA binding, was composed of oligo-L-lysine (K10) and HPMA. Melittin modified with cysteine at the C-terminus was conjugated to the polymers through the pyridyl disulfide pendant groups via disulfide exchange. The resulting pHgMelbHK10 copolymers are more membrane-lytic than melittin-free control polymers, and efficiently condensed plasmid DNA into salt-stable particles (~ 100–200 nm). The melittin-modified polymers transfected both HeLa and neuron-like PC-12 cells more efficiently than melittin-free polymers although toxicity associated with the melittin peptide was observed. Optimized formulations containing the luciferase reporter gene were delivered to mouse brain by intraventricular brain injections. Melittin-containing polyplexes produced about 35-fold higher luciferase activity in the brain compared to polyplexes without melittin. Thus, the melittin-containing block copolymers described in this work are promising materials for gene delivery to the brain.

Our report describes RAFT copolymerization of multiple species of active peptide monomers with N-(2-hydroxypropyl(methacrylamide) (HPMA) under aqueous conditions. Resulting statistical copolymers are narrowly disperse and with highly controlled molecular weight and composition. Side chain peptide-copolymers were synthesized using a DNA condensing peptide (K12), and an endosomal escape peptide (K6H5) that had been modified with an aminohexanoic linker and capped with methacrylamide vinyl on the NH2-terminus. Copolymers of HMPA-co-K12 and HPMA-co-K12-co-K6H5 efficiently condensed DNA into small particles that maintain size stability even in 150 mM salt solutions. With increasing peptide content, the peptide-based polymers demonstrated gene delivery efficiencies to cultured human cells that were comparable to linear polyethylenimine.

Several drug delivery designs combine synthetic drug carriers with covalently conjugated targeting moieties. Such modifications of monoclonal antibodies (mAb), or their Fab′ fragments, inevitably result in diminished affinity for their targeted tissue. In an attempt to overcome this limitation, high molecular weight, branched N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and conjugated with Fab′ fragments of the anti-CD20 antibody, 1F5. This produced multivalent conjugates with varying valency (amount of Fab′ per macromolecule) targeted to the B-cell antigen CD20. Evaluation of a multivalent effect was done by determining the apparent KD at low concentrations of conjugates, the Sips heterogeneity factor, a, and the binding enhancement factors of each construct. The results clearly indicated that multivalency could improve the affinity of the HPMA copolymer–Fab′ conjugates to that of unconjugated mAb.

Background/aims: Telemedicine offers potential to improve the accessibility and quality of diagnosis of retinopathy of prematurity (ROP). The aim of this study was to measure accuracy of remote image based ROP diagnosis by three readers using receiver operating characteristic (ROC) analysis. Methods: 64 hospitalised infants who met ROP examination criteria underwent two consecutive bedside procedures: dilated examination by an experienced paediatric ophthalmologist and digital retinal imaging with a commercially available wide angle camera. 410 images from 163 eyes were reviewed independently by three trained ophthalmologist readers, who classified each eye into one of four categories: no ROP, mild ROP, type 2 prethreshold ROP, or ROP requiring treatment. Sensitivity and specificity for detection of mild or worse ROP, type 2 prethreshold or worse ROP, and ROP requiring treatment were determined, compared to a reference standard of dilated ophthalmoscopy. ROC curves were generated by calculating values for each reader at three diagnostic cut-off levels: mild or worse ROP (that is, reader was asked whether image sets represented mild or worse ROP), type 2 prethreshold or worse ROP (that is, reader was asked whether image sets represented type 2 prethreshold or worse ROP), and ROP requiring treatment. Results: Areas under ROC curves ranged from 0.747-0.896 for detection of mild or worse ROP, 0.905-0.946 for detection of type 2 prethreshold or worse ROP, and 0.941-0.968 for detection of ROP requiring treatment. Conclusions: Remote interpretation is highly accurate among multiple readers for the detection of ROP requiring treatment, but less so for detection of mild or worse ROP.