Improvement of Cellular Uptake and Transfection Ability of pDNA Using α-Cyclodextrin-Polyamidoamine Conjugates as Gene Delivery System.

Polyamidoamine (PAMAM) dendrimers are a class of unique nanomaterials which attracted attention because of their extraordinary properties, such as highly branched structure and types of terminal primary groups. In addition, development in PAMAM chemical modification has broadened its biological application especially for drug and gene delivery. In this study, PAMAMs are covalently conjugated onto α-Cyclodextrin (α-CD) via amide bonds obtaining the starburst cationic polymers (CD-PG2). The chemical structure and composition of CD-PG2 was characterized by IH NMR. Physicochemical and biological properties of CD-PG2/pDNA polyplex were evaluated by agarose gel retardation, stability test against DNasecñ, MTT assay, DLS measurement, CLSM observation, LDH leakage test, cellular uptake route analysis and in-vitro cell transfection. Results showed that CD-PG2 can efficiently condense pDNA into nanoscale particles with a narrow size distribution, and protect pDNA form DNase I degradation. Compared with free PEI-25K and commercial product Lipofectamine2000, CD-PG2 shows excellent gene transfection efficiency without serum interference as well as relatively low cytotoxicity. Cellular uptake of CD-PG2/pDNA polyplex is mainly through CME and CvME route and further investigations demonstrate that α-CD can regulate CvME pathway to improve polyplex transfection behavior. In conclusion, CD-PG2 can be considered as a versatile tool for gene delivery, especially for gene transfer in-vivo.

Thiolated α-Cyclodextrin: The Invisible Choice to Prolong Ocular Drug Residence Time.

It was the aim of this study to develop cysteamine-conjugated α-cyclodextrin (α-CD) enabled to form disulfide bonds with cysteine-rich substructures of the ocular mucus layer to provide a prolonged residence time of incorporated drugs at the site of action. Cysteamine was covalently attached to oxidized α-CD via reductive amination. The resulting α-CD-cysteamine conjugates (α-CD-Cys) were characterized regarding the amount of free thiol groups attached to the oligomer backbone via Ellman's reagent; resazurin assay was conducted for cytotoxicity, and mucoadhesive properties were evaluated on porcine intestinal and ocular mucosal tissues. Furthermore, albino rabbits were used for assessing the irritation-masking effects of α-CD-Cys. Free thiol groups attached to the backbone were in the range of 558 ± 24-1143 ± 92 µmol/g. None of these α-CD-Cys unduly affected the viability of Caco-2 cells in a concentration of 0.5%. Mucoadhesive properties of α-CD-Cys were up to 32-fold improved compared to unmodified α-CD. Encapsulation of cetirizine into α-CD-Cys resulted in significantly reduced local ocular mucosal irritation of this model drug. According to these results, α-CD-Cys is a promising new tool to prolong drug residence time on the ocular mucosal surface.

Effects of dendrimer/cyclodextrin conjugates as gene transfer carriers on nitric oxide production from macrophages.

OBJECTIVES: The development of safe gene transfer carriers with high transfection efficiency, which does not affect the cell function, is a challenging issue. In this study, we examined the effects of α-cyclodextrin (α-CyD)/dendrimer conjugate (α-CDE (G3)) on nitric oxide (NO) production in murine macrophages J774.1 cells stimulated with toll-like receptors (TLR) ligands. METHODS: NO production from macrophages stimulated with TLR ligands was determined by the Griess method. Transfection efficiency of α-CDE (G3)/plasmid DNA (pDNA) complex was quantified by a luminometer. KEY FINDINGS: α-CDE (G3) significantly inhibited NO production from J774.1 cells stimulated with TLR ligands. α-CyD molecules in α-CDE (G3) had no effect on NO production. The inhibitory effect of α-CDE (G3) on NO production might be attributed to the dendrimer (G3). Increasing the degree of substitution (DS) of α-CyD in the α-CDE (G3) molecule was accompanied by a significant decrease in the inhibition of NO production. Furthermore, higher gene transfection efficiency of α-CDE (G3)/pDNA complex was observed upon increasing the DS of α-CyD. CONCLUSIONS: α-CDE (G3) with high DS value of α-CyD may be considered as a safe gene transfer carrier that does not adversely affect NO production from macrophages stimulated with TLR ligands.