Thermosensitive amphiphilic block copolymers self-assemble into micelles above their lower critical solution temperature in water, however, the micelles generally display mediocre physical stability. To stabilize such micelles and increase their loading capacity for chemotherapeutic drugs, block copolymers with novel aromatic monomers were synthesized by free radical polymerization of N-(2-benzoyloxypropyl methacrylamide (HPMAm-Bz) or the corresponding naphthoyl analogue (HPMAm-Nt), with N-(2-hydroxypropyl) methacrylamide monolactate, using a polyethylene glycol based macroinitiator. The critical micelle temperatures and critical micelle concentrations decreased with increasing the HPMAm-Bz/Nt content. The micelles of 30-50 nm were prepared by heating the polymer aqueous solutions from 0 to 50 °C and were colloidally stable for at least 48 h at pH 7.4 and 37 °C. Paclitaxel and docetaxel encapsulation was performed by mixing drug solutions in ethanol with polymer aqueous solutions and heating from 0 to 50 °C. The micelles had a drug loading capacity up to 34 wt % for docetaxel, which is among the highest loadings reported for polymeric micelles, with loaded micelle sizes ranging from 60 to 80 nm. The micelles without aromatic groups almost completely released loaded paclitaxel in 10 days, whereas the HPMAm-Bz/Nt containing micelles released 50% of the paclitaxel at the same time, which showed a better retention for the drug of the latter micelles. (1)H solid-state NMR spectroscopy data are compatible with π-π stacking between aromatic groups. The empty micelles demonstrated good cytocompatibility, and paclitaxel-loaded micelles showed high cytotoxicity to tumor cells. In conclusion, the π-π stacking effect introduced by aromatic groups increases the stability and loading capacity of polymeric micelles.

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Decationized polyplexes have previously shown unique features, especially regarding their excellent cytocompatibility and very low degree of nonspecific cellular uptake. In the present study, targeted disulfide cross-linked decationized polyplexes were composed of a core of disulfide cross-linked poly(hydroxypropyl methacrylamide) (pHPMA) stably entrapping plasmid DNA (pDNA) and a shell of poly(ethylene glycol) (PEG) decorated with folate molecules. Folate was used as targeting ligand because of its high binding affinity to its receptor, which is overexpressed in many tumors. Studies using folate receptor overexpressing cell lines (HeLa and OVCAR-3) showed significantly higher cell uptake for the folate-targeted decationized polyplexes, when compared to their nontargeted counterparts. On the contrary, for a nonexpressing folate receptor cell line (A549) similar uptake was observed for both targeted and nontargeted decationized polyplexes. Transfection studies using OVCAR-3 cells showed higher transfection efficiency for folate-targeted polyplexes, because of improved cellular uptake. Simultaneously, introduction of targeting moiety on polyplexes did not affect their good cytocompatibilty. The results reported in this paper demonstrate that coupling of folate to decationized polyplexes generates a potential system for targeted gene delivery.

Many polycation-based gene delivery vectors show high transfection in vitro, but their cationic nature generally leads to significant toxicity and poor in vivo performance which significantly hampers their clinical applicability. Unlike conventional polycation-based systems, decationized polyplexes are based on hydrophilic and neutral polymers. They are obtained by a 3-step process: charge-driven condensation followed by disulfide crosslinking stabilization and finally polyplex decationization. They consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). In the present paper the applicability of decationized polyplexes for systemic administration was evaluated. Cy5-labeled decationized polyplexes were evaluated for stability in plasma by fluorescence single particle tracking (fSPT), which technique showed stable size distribution for 48 h unlike its cationic counterpart. Upon the incubation of the polymers used for the formation of polyplexes with HUVEC cells, MTT assay showed excellent cytocompatibility of the neutral polymers. The safety was further demonstrated by a remarkable low teratogenicity and mortality activity of the polymers in a zebrafish assay, in great contrast with their cationic counterpart. Near infrared (NIR) dye-labeled polyplexes were evaluated for biodistribution and tumor accumulation by noninvasive optical imaging when administered systemically in tumor bearing mice. Decationized polyplexes exhibited an increased circulation time and higher tumor

The applicability of small interfering RNA (siRNA) in future therapies depends on the availability of safe and efficient carrier systems. Ideally, siRNA delivery requires a system that is stable in the circulation but upon specific uptake into target cells can rapidly release its cargo into the cytoplasm. Previously, we evaluated a novel generation of carrier systems ("decationized" polyplexes) for DNA delivery, and it was shown that folate targeted decationized polyplexes had an excellent safety profile and showed intracellular triggered release upon cell specific uptake. Targeted decationized polyplexes consist of a core of disulfide cross-linked poly(hydroxypropyl methacrylamide) (pHPMA) stably entrapping nucleic acids and a shell of poly(ethylene glycol) (PEG) decorated with folate molecules. In the present study, the applicability of folate targeted decationized polyplexes for siRNA delivery was investigated. This required optimization of the carrier system particularly regarding the cross-linking density of the core of the polyplexes. Stable and nanosized siRNA decationized polyplexes were successfully prepared by optimizing the cross-link density of their core. Upon incubation in human plasma, a significant portion of siRNA remained entrapped in the decationized polyplexes as determined by fluorescence correlation spectroscopy (FCS). When tested in a folate receptor overexpressing cell line stably expressing luciferase, Skov3-luc, sequence specific gene silencing was observed. As expected, neither interference on the intrinsic luciferase expression nor on the cell metabolic activity (determined by XTT) was induced by the free-polymer or the siRNA polyplexes. In conclusion, targeted decationized polyplexes are safe and stable carriers that interact with the targeted cells and rapidly disassemble upon cell entry making them promising siRNA delivery systems.

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Purpose. The aim of this study was to investigate the in vitro gene delivery efficiency of poly[N-(2-aminoethyl)ethylene-imine](PAEEI), a polymer with a linear Polyethyleneimine (LPEI) backbone and with aminoethyl side groups that has two protonatable nitrogen atoms per monomer unit instead of one as in LPEI (an established gene delivery polymer). Method. PAEEI (Mn=4.5 kDa, Mw= 10 kDa) was synthesized by ring-opening polymerization of N-(2-(1’-aziridino)ethyl)formamide followed by hydrolysis of the amide groups.  The buffering capacity of the resulting polymer was determined by acid-base titration and consequently the percentage of the protonated nitrogen atoms was calculated. Polyplexes were prepared separately in buffers with different ionic strength including Hepes buffered saline (150 mM NaCl) and Hepes buffered glucose (5% glucose) and their zeta-potential, hydrodynamic diameter and colloidal stability were measured. Transfection activity (and toxicity in Hela cells) of the polyplexes were done in HeLa, CHO and HEK293T cells. Cell incubations with polyplexes were done both in the presence and absence (HeLa cells) of serum. Results. PAEEEI showed two times more buffering capacity than LPEI. PAEEI-based Polyplexes had about the same size and zeta-potential as those of LPEI, with a higher colloidal stability in saline buffer in continuous particle size measurement. Their transfection activity was slightly higher than 22-kDa LPEI polyplexes whereas their toxicity profiles were similar in cell lines studied. The PAEEI polyplexes showed gene expression activity both in the presence and absence of serum. Conclusion. Paying attention to the fact that LPEI molecules with smaller sizes than 22 kDa  show less transfection efficiency than LPEI 22, the effect of smaller size of PAEEI (10 kDa) on the gene delivery efficiency was compensated by its higher buffering capacity due to carrying more protonatable nitrogen per monomeric unit comparing with LPEI (22 kDa). Having slightly higher transfection efficiency and better colloidal stability than PEI-based systems, PAEEI is an attractive candidate for future in vivo gene delivery studies.This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.