Carbon
dots (CDs) have become the focus of many studies due to
their outstanding optical properties and good biocompatibility. We
investigated their potential application to produce a smart and highly
efficient yet nontoxic nanovector for gene delivery. This was achieved
by conjugating PEI1.8k-functionalized CDs (synthesized
by one-step microwave-assisted pyrolysis) with arginine-disulfide
linkers to produce CD-PEI1.8k-Arg nanoparticles. This nanovector
could deliver p-CRISPR (9.3 kb) into different types of cell lines
with higher efficiency compared to native PEI1.8k or PEI25k. CD-PEI1.8k-Arg also maintained its outstanding
transfection efficiency at a high serum concentration and low p-CRISPR
dose, compared to PEI25k, which was ineffective under those
conditions. Additionally, CD-PEI1.8k-Arg could knock out
the GFP gene with great efficiency by delivering the required components
of CRISPR/Cas9, including a plasmid encoding Cas9, sgRNA targeting
GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) into the HEK 293T-GFP
cells. Moreover, the nanoparticles showed potential for the local
delivery of p-CRISPR into brain tissue. The remarkable properties
of CD-PEI1.8k-Arg could enable the development of a safe,
highly efficient gene-delivery nanovector for the treatment of various
diseases in the near future.
Safe
and efficient delivery of CRISPR/Cas9 systems is still a challenge.
Here we report the development of fluorescent nitrogen- and zinc-doped
carbon dots (N–Zn-doped CDs) using one-step microwave-aided
pyrolysis based on citric acid, branched PEI25k, and different
zinc salts. These versatile nanovectors with a quantum yield of around
60% could not only transfect large CRISPR plasmids (∼9 kb)
with higher efficiency (80%) compared to PEI25k and lipofectamine
2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with
the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N–Zn-doped
CDs exhibited good transfection efficiency even at low plasmid doses
and in the presence of 10% fetal bovine serum (FBS). Moreover, these
nanovectors demonstrated excellent efficiency in GFP gene disruption
by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well
as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N–Zn-doped
CDs with remarkable photoluminescence properties and high transfection
efficiency in the delivery of both CRISPR complexes and mRNA provide
a promising platform for developing safe, efficient, and traceable
delivery systems for biological research.
An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8k in the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5–10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid and in vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.
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