Design, synthesis, and optimization of nanostructured calcium phosphates (NanoCaPs) and natural polymer based 3-D non-viral gene delivery systems.

摘要

Sustained delivery of therapeutic genes from a three-dimensional (3-D) scaffold and subsequent gene expression capable of triggering the regeneration of damaged tissues is a tissue engineering strategy that has been gaining increased attention. Nanostructured calcium phosphates (NanoCaPs) are biocompatible and non-toxic biomaterials. Furthermore, their efficient transfection in vitro have rendered them attractive gene delivery carriers compared to other viral- or lipid-based agents that tend to be immunogenic or cytotoxic, leading to undesirable responses when utilized above a critical threshold. However, NanoCaPs are typically characterized by variable transfection and short shelf life due to particle aggregation. A viable solution to this problem is the incorporation of NanoCaPs into 3-D scaffolds. The main objectives of this research are therefore two-fold: (1) Examination of the potential of achieving optimized transfection of NanoCaPs via anionic substitution and (2) high throughput synthesis and screening of non-viral gene delivery systems (GDS) comprised of naturally-derived polymers as scaffolds containing NanoCaPs gene carriers.;Results indicated that in addition to the excellent transfection levels exhibited by NanoCaPs in vitro, an additional 20-30% increase was observed for NanoCaPs with 10-25 mol% anion substitution. In contrast, high anion substitution (>60%) yielded a drastic decline in transfection. Structural characterizations verified successful anion substitution with a noticeable increase in lattice parameters indicative of an expanded unit cell due to ionic substitution. All of the anion substituted calcium phosphates exhibited the primary phase of hydroxyapatite.;For the first time, GDS composed of various concentrations of alginate (AA), fibronectin (FN), and NanoCaPs-DNA complexes were demonstrated. The presence of AA and FN was effective in immobilizing NanoCaPs and reducing the aggregation. High throughput synthesis and screening experiments showed excellent cell proliferation in the presence of AA, and the incorporation of FN improved cell attachment on AA. Moreover, AA-based GDS showed optimized transfection following 3 weeks of storage, extending the shelf life of NanoCaPs.;Also for the first time, GDS composed of various concentrations of fibrin (F), gelatin (G), and NanoCaPs-DNA complexes were demonstrated. The presence of F and G also resulted in the uniform immobilization and distribution of NanoCaPs, reducing the aggregation. It was also established that the concentration as well as the ratio of F and G played a significant role in ultimately determining the transfection. Both luciferase and green fluorescent protein (GFP) transfection revealed a two-step release mechanism, with an early burst release upon the dissolution of G followed by a subsequent delayed and sustained release corresponding to the degradation of F. This composite GDS can therefore be tailored to achieve optimal gene transfection, rendering it viable for various therapeutic applications.