Transdermal delivery of CRISPR/Cas9-mediated melanoma gene therapy via polyamines-modified thermosensitive hydrogels

Abstract

The main obstacles to the clinical application of the CRISPR/Cas9 system are off-target effects and low delivery efficiency. There is an urgent need to develop new delivery strategies and technologies. Three types of in situ injectable hydrogels with different electrical properties were created to find the most secure and efficient sustained-release drug delivery system. After in vitro and in vivo comparisons, we found that the positively charged hydrogels had higher cellular uptake, stronger gene editing efficiency, greater cytotoxicity, longer tumor accumulation, and better anti-tumor efficacy than negatively charged and neutral hydrogels. We designed single guide RNA targeting the Y-box binding protein 1 (YB-1) gene and then used it to create a ribonucleoprotein complex with Cas9 protein. Doxorubicin was co-encapsulated into this positively charged hydrogel to create a co-delivery system. By knocking down YB-1, the expression of YB-1 was reduced, inhibiting the growth and migration of melanoma cells. The strategy of combining YB-1 gene editing and intratumoral injection enhanced the therapeutic effect of doxorubicin while reducing side effects.

Keywords: CRISPR/Cas9; Hydrogel; Intratumoral injection; Polyamines; YB-1.

Fig. 1

Schematic of the preparation, delivery, and intracellular fate of Psh@DOX@RNP

Fig. 2

Preparation, characterization and comparison of three hydrogels. (a) Structures of polyamines utilized in the study. (b) Schematic representation of micelle formation by Poloxamer at elevated temperatures. (c) Zeta potential of different hydrogels. Column 1, Nuh; Column 2–4, Nuh@0.05%HA, Nuh@0.1%HA, Nuh@0.15%HA; Column 5–7, Nuh@0.05%PA, Nuh@0.1%PA, Nuh@0.15%PA. (d) SEM photographs of the Psh. (e) Psh@DOX is a fluxible liquid at 4 °C and a gel state at 37 °C. (f) Subcutaneous status of Psh@DOX at different time points. (g) In vitro release of Psh@DOX. (h) In vitro release of Psh@FITC-BSA. (i) Evaluation of haemolysis at different concentrations of Nuh. (j) Evaluation of haemolysis at different concentrations of Psh. (k) 2% agarose gel image of RNP-loaded hydrogel preparations. sgRNA was used to determine the encapsulation rate of RNP in hydrogels. (l) 2% agarose gel image of Psh@RNP. Data are presented as the mean ± s.d. (n = 3 biological replicates per group)

Fig. 3

Cellular uptake of DiD and FITC mediated by three types of hydrogels. (a) Confocal microscope images of B16F10 cells after 6 h incubation with Aq@DiD@FITC-BSA, Nuh@DiD@FITC-BSA, Ngh@DiD@FITC-BSA, Psh@DiD@FITC-BSA and Lip2000@DiD@FITC-BSA. Cell nucleus was counterstained with 4,6-diamino-2-phenyl indole (DAPI) (blue). Quantitative analysis of (b) DiD and (c) FITC in B16F10 cells. (d) Endocytosis of Aq@DiD@FITC-BSA, Nuh@DiD@FITC-BSA, Ngh@DiD@FITC-BSA, Psh@DiD@FITC-BSA or Lip2000@DiD@FITC-BSA by B16F10 cells, detected by flow cytometry

Fig. 4

Pharmaceutical effect of Psh@DOX@RNP on tumor cell B16F10 and non-tumor cell 293T. (a) The toxicity of Psh, Ngh and Nuh on 293T cells at different incubation time. (b) The toxicity of Psh, Ngh and Nuh on B16F10 cells at different incubation time. The viability of B16F10 cells after being treated by (c) free DOX; (d) Ngh@DOX, Nuh@DOX, Psh@DOX; (e) Psh, Psh@Cas9, Psh@RNP (Cas9/sgRNA at molar mass ratio of 1:1, 1:2, 1:3, 1:4, 1:5) and (f) Psh, Psh@DOX, Psh@Cas9, Psh@RNP, Aq@DOX@RNP, Psh@DOX@RNP(GFP), Psh@DOX@RNP. (g) Fluorescence microscopy images of PI (red) and calcein AM (green) cells after different treatments. (h) Apoptosis induced by various treatments to B16F10 cells using flow cytometry. Data are presented as the mean ± s.d. (n = 3 biological replicates per group)

Fig. 5

In vitro gene editing efficiency of Psh@DOX@RNP. (a) Endonuclease activity of RNPs (composed of different sgRNAs) on PUC18@YB-1 plasmid in vitro. (b) Endonuclease activity of RNP (Cas9/sgRNA at molar mass ratio of 1:1, 1:2, 1:3, 1:4, 1:5) on PUC18@YB-1 plasmid in vitro. (c) T7EI assay of B16F10 cells treated with PBS, Aq@RNP, Nuh@RNP, Ngh@RNP and Psh@RNP. (d) The mRNA level of YB-1 was analysed by qRT-PCR. Data are presented as the mean ± s.d. (e) Representative images of the wound healing assays in B16F10 cells from 0 h to 72 h. (f) Fluorescence images of 293T-GFP cells treated with different preparations [PBS, Ngh@RNP(GFP), Nuh@RNP(GFP), Psh@RNP(GFP), Lip2000@RNP(GFP)]. (n = 3 biological replicates per group)

Fig. 6

Sustained release and accumulation of RNP and DOX delivered by Psh in melanoma mouse models. In vivo fluorescence imaging of (a) DiD and (b) FITC in mice at different time points after intratumoral injection of PBS@DiD@FITC-BSA, Nuh@DiD@FITC-BSA, Ngh@DiD@FITC-BSA, or Psh@DiD@FITC-BSA. Quantification of (c) DiD and (d) FITC in mice by in vivo fluorescence imaging. (e) In vivo fluorescence images of isolated tumors from mice after 8 days injection. Cell nucleus was counterstained with DAPI (blue). (f, g) Quantitative analysis of drug accumulation in tumors. Data are presented as the mean ± s.d. (n = 3 biological replicates per group)

Fig. 7

In vivo anti-tumor efficiency of Psh@DOX@RNP. (a) Treatment scheme. (b) Photographs of tumor dissected from C57BL/6 mice treated with PBS, Aq@DOX@RNP, Psh@DOX, or Psh@DOX@RNP. (c) Tumor weight of the mice. (d) Tumor growth curve of the mice after different treatments. (e) Body weight of C57BL/6 mice after treatment. (f) H&E stained sections and (g) Ki67 Immunohistochemical staining of isolated tumors. (h) TUNEL analysis in tumor sections after different treatments. Data are presented as the mean ± s.d. (n = 6 biological replicates per group)

Fig. 8

In vivo gene editing efficiency of Psh@DOX@RNP. (a) Immunofluorescence analysis of Cas9 (green), YB-1(red) expression. (b) Immunohistochemical analysis of P53 expression in tumor tissue sections. Quantitative analysis of (c) Cas9 protein and (d) YB-1 expression. (e) Quantitative analysis of the number of P53-positive cells. (f) T7EI assay of the isolated tumor tissue. (g) The mRNA of YB-1 in tumor tissues quantified by qRT-PCR. (h) T7EI assay of the isolated heart, liver, spleen, lung and kidney. Data are presented as the mean ± s.d. (n = 6 biological replicates per group)