Tatu Lajunen

DNA origami nanoparticles (DONs) are attractive nanocarriers of controllable size, shape, and addressability that have potential for treating eye diseases by overcoming ocular barriers. However, suboptimal physiological stability and poor cell uptake due to the negative charge may limit their use. Previous reports show that electrostatic complexation of DONs with cationic PEG‐oligolysine block copolymers like PEG5K‐K10 can improve structural integrity and promote cell internalization. Here, we investigated a dual approach of PEG5K‐K10 coatings and PL3‐targeting peptides to improve uptake of 24‐helix bundle (24HB) DONs into Y‐79 retinoblastoma cells. Uptake studies revealed that PEG5K‐K10 was essential for DON uptake in Y‐79 cells, as uptake only occurred upon exceeding a distinct PEG5K‐K10 amount. Longer exposure times or increased polymer amounts improved cell association. However, no beneficial effect of PL3 was observed. While free PEG5K‐K10 reduced cell viability at higher concentrations (IC ₅₀ 36.8 µM), coated DONs were well‐tolerated. Furthermore, single particle tracking in ex vivo porcine eyes revealed comparable vitreal mobility for uncoated and coated 24HB, with a slight decrease at higher coating amounts. Our findings highlight that PEG5K‐K10 can enhance ocular cell uptake without limiting nanoparticle diffusivity in the vitreous and support further optimization of DONs for ocular drug delivery.

DNA origami nanoparticles (DONs) are attractive nanocarriers of controllable size, shape and addressability that have potential for treating eye diseases by overcoming ocular barriers. However, suboptimal physiological stability and poor cell uptake due to the negative charge may limit their use. Previous reports show that electrostatic complexation of DONs with cationic PEG-oligolysine block-copolymers like PEG5K-K10 can improve structural integrity and promote cell internalization. Here, we investigated a dual approach of PEG5K-K10 coatings and PL3 targeting peptides to improve uptake of 24-helix bundle (24HB) DONs into Y-79 retinoblastoma cells. Uptake studies revealed that PEG5K-K10 was essential for DON uptake in Y-79 cells, as uptake only occurred upon exceeding a distinct PEG5K-K10 amount. Longer exposure times or increased polymer amounts improved cell association. However, no beneficial effect of PL3 was observed. While free PEG5K-K10 reduced cell viability at higher concentrations (IC ₅₀ 36.8 µM), coated DONs were well-tolerated. Furthermore, single particle tracking in ex vivo porcine eyes revealed comparable vitreal mobility for uncoated and coated 24HB, with a slight decrease at higher coating amounts. Our findings highlight that PEG5K-K10 can enhance ocular cell uptake without limiting nanoparticle diffusivity in the vitreous, and support further optimization of DONs for ocular drug delivery.

Background: Sirtuin-1 (SIRT1), a histone deacetylase enzyme expressed in ocular tissues with intracellular localization, plays a critical protective role against various degenerative ocular diseases. The link between reduced SIRT1 levels and diabetic retinopathy (DR) has prompted the exploration of natural therapeutic compounds that act as SIRT1 agonists. Curcumin (CUR), which has been shown to upregulate SIRT1 expression, is one such promising compound. However, effective delivery of CUR to the deeper ocular tissues, particularly the retina, remains a challenge due to its poor solubility and limited ocular penetration following topical administration. Within this context, the development of self-nanoemulsifying drug delivery systems (SNEDDS) for CUR topical ocular delivery represents a novel approach. Methods: In accordance with our prior research, optimized SNEDDS loaded with CUR were developed and characterized post-reconstitution with simulated tear fluid (STF) at a 1:10 ratio, showing suitable physicochemical and technological parameters for ocular delivery. Results: An entrapment efficiency (EE%) of approximately 99% and an absence of drug precipitation were noticed upon resuspension with STF. CUR-SNEDDS resulted in a better stability and release profile than free CUR under simulated ocular conditions. In vitro analysis of mucoadhesive properties revealed that CUR-SNEDDS, modified with a cationic lipid, demonstrated enhanced interactions with mucin, indicating the potential for improved ocular retention. Cytotoxicity tests demonstrated that CUR-SNEDDS did not affect the viability of human corneal epithelial (HCE) cells up to concentrations of 3 μM and displayed superior antioxidant activity compared to free CUR in an oxidative stress model using retinal pigment epithelial (ARPE-19) cells exposed to hydroquinone (HQ). Cell uptake studies confirmed an enhanced accumulation of CUR within the retinal cells following exposure to CUR-SNEDDS compared to neat CUR. CUR-SNEDDS, at lower concentrations, were found to effectively induce SIRT1 expression. Conclusions: The cytocompatibility, antioxidant properties, and enhanced cellular uptake suggest that these developed systems hold promise as formulations for the delivery of CUR to the retina.

Sirtuin-1 (SIRT1), a histone deacetylase enzyme expressed in ocular tissues with intracellular localization, plays a protective role against multiple ocular diseases such as retinal degeneration, cataracts, and optic neuritis. The correlation between diabetic retinopathy (DR) and decreased levels of SIRT1 has stimulated investigation into natural therapeutic compounds that function as agonists. Curcumin (CUR), which upregulates SIRT1 protein expression, has been identified as a molecule with agonist properties. However, the challenge lies in delivering CUR effectively to the deeper layers of the eye, as retinal, due to its poor solubility and inadequate ocular penetration after topical administration. Within this context, the development of self-nanoemulsifying drug delivery systems (SNEDDS) for CUR topical ocular delivery can represent a novel approach. In accordance with our prior research, optimized SNEDDS loaded with CUR were characterized post-reconstitution with simulated tear fluid (STF) at a 1:10 ratio.Optimal parameters were achieved, ensuring mean globule size<50 nm, PDI <0.2, emulsification time 95%), viscosity, pH, and osmolarity close to physiological values. An entrapment efficiency (EE%) of approximately 99% and absence of drug precipitation were noted upon resuspension with STF. CUR loaded in these systems exhibited better stability than free CUR up to 1 week exposed to different conditions and demonstrated burst release following sustained release under simulated ocular conditions. Mucoadhesion studies were performed in STF for both CUR-SNEDDS and CUR-SNEDDS positively charged with dimethyldidodecylammonium bromide (DDAB). SNEDDS exhibited no adverse effects on the viability of human corneal epithelial (HCE) cells up to 3 μM and demonstrated superior antioxidant activity compared to neat CUR on retinal cells (ARPE-19) exposed to hydroquinone. Cell uptake studies confirmed enhanced accumulation of CUR within retinal cells (around 20% of CUR internalized) following exposure to CUR-SNEDDS compared to neat CUR (0-0.5% of CUR internalized). CUR-SNEDDS, at lower concentrations, were found to effectively induce SIRT1 expression. The cytocompatibility, antioxidant properties, and enhanced cellular uptake suggest that these developed systems hold promise as formulations for the delivery of CUR to the retina.

Doxorubicin-loaded DNA origami required additional Mg²⁺ to be stable in cell medium and vitreous at 37 °C. In retinal epithelial cells (ARPE-19), they were well-tolerated, but limited in their uptake.

RNA-based therapeutics, including siRNA, have obtained recognition in recent years due to their potential to treat various chronic and rare diseases. However, there are still limitations to lipid-based drug delivery systems in the clinical use of RNA therapeutics due to the need for optimization in the design and the preparation process. In this study, we propose adaptive focused ultrasound (AFU) as a drug loading technique to protect RNA from degradation by encapsulating small RNA in nanoliposomes, which we term nanoplexes. The AFU method is non-invasive and isothermal, as nanoplexes are produced without direct contact with any external materials while maintaining precise temperature control according to the desired settings. The controllability of sample treatments can be effectively modulated, allowing for a wide range of ultrasound intensities to be applied. Importantly, the absence of co-solvents in the process eliminates the need for additional substances, thereby minimizing the potential for cross-contaminations. Since AFU is a non-invasive method, the entire process can be conducted under sterile conditions. A minimal volume (300 μL) is required for this process, and the treatment is speedy (10 min in this study). Our in vitro experiments with silencer CD44 siRNA, which performs as a model therapeutic drug in different mammalian cell lines, showed encouraging results (knockdown > 80%). To quantify gene silencing efficacy, we employed quantitative polymerase chain reaction (qPCR). Additionally, cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) techniques were employed to capture images of nanoplexes. These images revealed the presence of individual nanoparticles measuring approximately 100–200 nm in contrast with the random distribution of clustered complexes observed in ultrasound-untreated samples of liposome nanoparticles and siRNA. AFU holds great potential as a standardized liposome processing and loading method because its process is fast, sterile, and does not require additional solvents.