Abstract
Drug delivery to the brain is highly hindered by the presence of the blood–brain barrier (BBB), which prevents the entry of many potential drugs/biomolecules into the brain. One of the current strategies to achieve gene therapy for neurodegenerative diseases involves direct injection of a viral vector into the brain. There are various disadvantages of viral vectors, including limitations of cargo size and safety concerns. Nanomolecules, such as dendrimers, serve as an excellent alternative to viral delivery. In this study, as proof-of-concept, we used a surface-modified dendrimer complex and delivered large plasmids to cells in vitro and in vivo in healthy rats via intracranial injection. The dendrimers were biodegradable by chemicals found within cells and toxicity assays revealed that the modified dendrimers were much less toxic than unmodified amine-surface dendrimers. As mentioned in our previous publication, these dendrimers with appropriately modified surfaces are safe, can deliver large plasmids to the brain, and can overcome the cargo size limitations associated with viral vectors. The biocompatibility of this dendritic nanomolecule and the ability to finely tune its surface chemistry provides a gene delivery system that could facilitate future in vivo cellular reprograming and other gene therapies.
Highlights
Effective delivery of nucleic acids, such as clustered regularly interspaced short palindromic repeat associated proteins (CRISPR/Cas9), transcription activator-like effectors (TALEs), zinc finger sequences (ZFNs), and small interfering RNA, into cells is a critical requirement for cell reprogramming, gene therapy, and vaccine development
Nucleic acids are readily degraded by nucleases that are ubiquitous in the body
We developed a much simpler de novo method for the synthesis of G4-90/10, as previously mentioned in our manuscript [28], based on the original, well-established PAMAM dendrimer synthesis scheme used for synthesis of pure-surface dendrimers [27] (Figure 2)
Summary
Effective delivery of nucleic acids, such as clustered regularly interspaced short palindromic repeat associated proteins (CRISPR/Cas9), transcription activator-like effectors (TALEs), zinc finger sequences (ZFNs), and small interfering RNA (siRNA), into cells is a critical requirement for cell reprogramming, gene therapy, and vaccine development. The G4 amine-terminated dendrimer is highly effective in packaging and delivering nucleic acids into cells, it is more toxic than the lower generation dendrimers due to the high positive charge density from the 64 primary amines on its surface To circumvent this issue, a nanomolecule with a well-defined and reproducible number of surface amines was designed. The current strategy of reducing dendrimer toxicity involves decreasing the number of surface amines by attaching various chemicals, such as polyethylene glycol (PEG), cyclodextrins, glucocorticoids, methyl groups, amino acids, and several others [19,20,21,22,23,24,25] This strategy requires additional steps of synthesis and purification, complex protection/deprotection steps, is difficult to control, and yields heterogeneous mixtures of dendrimers. The percentages of surface amines were determined by 2,4,6-trinitrobenzene sulfonic acid assay (TS-28997 Thermo Fisher Scientific, Waltham, MA, USA), using glycine as the standard to prepare the calibration curve
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