Background: CRISPR/ Cas9 is one of the most powerful among the approaches being developed to rescue fundamental causes of gene-based inheritable diseases. Several strategies for delivering such genome editing materials have been developed, but the safety, efficacy over time, cost of production, and gene size limitations are still under debate and must be addressed to further improve applications. Sodium alginate is frequently used as the model encapsulation matrix for bioactive ingredients in the field of drug and gene delivery due to its safety. Objective: To encapsulate CRISPR/Cas9 plasmid DNA in alginate nanocarrier to perform genome editing. Methodology: alginate nanoparticles loaded with two CRISPR pDNA were fabricated using electrospray method. Both formulation and process were optimised. Chitosan-, Arabic gum- and PEG-coated CRISPR-loaded alginate nanoparticles were fabricated and characterised. CRISPR-loaded alginate nanoparticles physicochemical properties were compared to the surface-modified nanoparticle properties. The influence of surface modification of nanoparticles on their interaction with cell was studied in regard to cellular uptake, cytotoxicity, transfection efficiency, and genome editing. Results: Using electrospray, a nanoparticle carrier was developed to deliver CRISPR pDNA into HepG2 cells. The nanoparticles size was approximately 230 nm, with an encapsulation efficiency of 99%. Release study revealed that over one-third of the pDNA was released within the first 24 h. In vitro experiments conducted with HepG2 cells demonstrated that after 48 h of treatment with the CRISPR-loaded alginate nanoparticles, the particles were not toxic. CRISPR-loaded alginate nanoparticles mediated 1.5-folds more efficient transfection than a commercially available cationic liposome-based transfection reagent. However, their indel efficiency was 3.4-folds lower than the transfection reagent. The surface coating highly affected the nanoparticles physicochemical properties, consequently, their safety and efficiency in delivering the plasmid. CS CRISPR ALG NPs showed mean size and zeta potential of 377 nm and 33.67 mV, respectively. Over 90% encapsulation efficiency was achieved while protection payload from serum. The tests revealed that the nanoparticles were cytocompatible and successfully introduced the Cas9 transgene in HepG2 cells. CS CRISPR ALG NPs-transfected HepG2 was able to edit its target plasmid by introducing double-strand break (DSB) in GFP gene, 18.26-folds higher than CRISPR ALG NPs. Conclusions: In this work, plasmids for the CRISPR/Cas9 system were encapsulated in alginate nanoparticles and were shown to induce expression of Cas9 and perform a genome editing in HepG2 cells in vitro. Chitosan-coated CRISPR-loaded alginate nanoparticles revealed the best results with high plasmid protection, sustained release and high indel efficiency. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR/Cas9 system.

Background: Thymoquinone (TQ) has been known to have various therapeutic benefits but it is prone to fast degradation. TQ exhibited anticancer effect in several cancer cell lines. Encapsulation of TQ enhances its stability and provides a tool for targeting to the cancer tissues. Objective: To prepare and optimize TQ- poly-lactide-co-glycolide (PLGA) nanoparticle (NP) formulation and evaluate it in A375 cells for cell uptake and cytotoxicity. Methodology: TQ- PLGA NP formulation was prepared using solvent evaporation method and TQ quantification method was validated using HPLC. The total of 6 factors affecting the formulation (chitosan concentration (A), ratio of co-stabilizer (tween 80: polyvinyl alcohol (B), stabilizer concentration (C), ratio of co-solvents (ethyl acetate: dichloromethane) (D), sonication power (E) and sonication time (F), were screened using fractional factorial design. The screened factors were reduced to 3 most significant (A, C and E) affecting critical output responses of particle size, zeta potential and encapsulation efficiency. An optimized TQ-PLGA NP was prepared by altering these significant factors using full factorial design and targeted responses was acquired and it was proceed for cell uptake studies by flow cytometer and fluorescence microscope; and cell cytotoxicity studies by MTT assay. Results and discussion: Optimized TQ-PLGA NP formulation was prepared using 1.02 % w/v chitosan, 1.14% w/v stabilizer and 31% of sonication power. The rest of the parameters were fixed as 1:3 oil to aqueous phase ratio, 1:5 primary emulsion to dispersion medium ratio, 3% w/v PLGA concentration and 75:25 co-solvent ratios of ethyl acetate and dichloromethane. TQ-PLGA NP exhibited particle size of 147.2 ± 0.4 nm; polydispersity index (PDI) of 0.142 ± 0.017; zeta potential of 22.1 ± 1.1; encapsulation efficiency of 96.81 ± 0.05 %; biphasic release of 56.7 ± 1.3% (24 h) and 69.7 ± 1.3% within 1 week. The optimized formulation was aggregated in powdered form but stable in the suspension form up for 10 days. The formulation exhibited lower glass transition of PLGA below 37°C suggesting a plasticizing effect of TQ and other ingredients which contributes to the overall rapid release. TQ in methanol and TQ-PLGA NP in complete growth media were tested for stability. TQ degraded by 18% (24 h) and 77.0% (48 h), particle size remains stable in 24 h, significant changes occurred after 48 h (p < 0.05), PDI changes were significant post 24 h and 48 h (p < 0.05), significant changes on zeta potential were observed on higher concentration of TQ-PLGA NPs (p < 0.05). The highest intracellular uptake was at 1.0 mg/mL NP concentration with time-dependent cell uptake up to 24 h. Post 24 h treatment, the IC50 of TQ-PLGA NP and TQ-solution was calculated to be 4.428 mg/mL and 91.71 µg/mL respectively. At 48 h treatment, IC50s of TQ-PLGA NP was calculated to be 7.981 mg/mL and 58.657 mg/mL respectively. Conclusion: Optimized TQ-PLGA NP was formulated and it showed a promising cytotoxic effect in A375 cells. TQ in nanoparticle formulation has a potential use as anticancer and worth a further study in animal models.