Nucleic Acid Delivery Excipients

Carvalho BG, et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2024, 700, 134795.

Cationic liposomes can be used to prepare cationic lipid-polymer hybrid nanoparticles (cLPHNPs), which are also known as lipopolyplexes, and have been developed as a promising alternative to nonviral vectors. These core/shell structures aim to address the limitations associated with traditional cationic liposomes and biodegradable polymeric nanoparticles (NPs).

Preparation of Cationic Liposomes

Cationic liposomes (CLs), composed of EPC/DOPE/DOTAP in a 2:1:1 molar ratio, were prepared using the ethanol injection method with slight modifications. Lipids were dissolved in anhydrous ethanol to a final stock solution concentration of 400 mM. To produce CLs at 8 mM, a 500 μL glass syringe filled with the stock solution was used to feed a 25 mL cylindrical tank reactor with four baffles containing sterile water. A syringe pump fed the tank at a flow rate of 1.48 mL/min. The colloidal dispersion was formed through continuous stirring (6000 rpm for 15 minutes) using a digital disperser. The liposomal dispersion was then downsized using an extruder with a 100 nm polycarbonate membrane, repeated five times under 12 kgf/cm² nitrogen pressure. All samples were stored at 4 °C.

Preparation of Polyplexes and Lipopolyplexes

The theoretical charge ratio of pDNA/PEI complexes was calculated based on the molar ratio of nitrogen in bPEI to phosphate in pDNA per nucleotide, with 1 μg of pDNA equating to 3 nmol of phosphate. Polyplexes were formed through electrostatic interactions by mixing equal volumes of pDNA with bPEI in ultrapure water (18.2 MΩ.cm). The appropriate amount of bPEI was added to pEGFP-N3 depending on their N/P ratios (0.4, 0.6, 1, and 2), mixed by gentle pipetting, and incubated for 30 minutes at room temperature.

cLPHNPs were prepared by mixing anionic polyplexes (bPEI-pDNA, ratio 0.4) with (i) the previously prepared EPC/DOPE/DOTAP CLs dispersion (8 mM) through thorough pipetting, and (ii) EPC/DOPE/DOTAP lipid suspension (8 mM) through pipetting and continuous vortexing (600 rpm). In both cases, the final lipid concentration was adjusted to 1.5 mM, and the cLPHNPs dispersion was agitated for one minute.

Chen J, et al. Nanotoday, 2024, 57, 102335.

The hybrid nanoparticles DOTAP-hNPs composed of cationic lipid 2-dioleoyl-3-trimethylammonium-propane (DOTAP) and polymer poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG-b-PLGA) can effectively induce immunogenic cell death (ICD) of tumor cells and produce tumor antigens in situ.

The preparation method of DOTAP-hNPs is as follows:

Positively charged DOTAP-hNPs were produced by a single emulsification solvent evaporation method. First, 5 mL sterile water was added to 0.5 mL chloroform phase containing 25 mg of mPEG5k-b-PLGA11k and 4 mg DOTAP, and then the mixture was emulsified by repeated ultrasonication for 2 min (80 W, 10 seconds on, 2 seconds off) in a water-ice bath using ultrasonic Processor. Next, nanoparticles were formulated after the chloroform was removed under vacuum in a rotary evaporator RV10.

Chen X, et al. Biochemical and Biophysical Research Communications, 2024, 719, 150100.

Polyene phosphatidylcholine (PPC) is a crucial drug for liver repair and its potential role in promoting fracture healing warrants investigation.

Materials and Methods

A rat model of tibial fracture was developed using the modified Einhorn model method. X-rays were employed to monitor the progression of fracture healing. Ossification and angiogenesis at the fracture site were analyzed through Safranin O/fast green staining and CD31 immunohistochemistry. To assess whether PPC directly affects angiogenesis, human umbilical vein endothelial cells (HUVECs) were used in various assays, including MTT, wound healing, Transwell migration, and tube formation assays. Additionally, RT-qPCR and Western blot analyses were conducted to investigate the underlying mechanisms.

Results

The results revealed that PPC significantly reduced the apparent recovery time of mobility in rats. PPC treatment markedly enhanced the formation of cartilage callus, endochondral ossification, and angiogenesis at the fracture site. In vitro experiments showed that PPC increased the proliferative viability of HUVECs, enhanced their wound healing capabilities, improved their membrane penetration in the Transwell apparatus, and boosted tube formation.

At the molecular level, PPC significantly upregulated the transcription of VEGFA, VEGFR2, PLCγ, RAS, ERK1/2, and MEK1/2. Protein analysis indicated a notable increase in the expression of VEGFA, VEGFR2, MEK1/2, and ERK1/2 proteins.

Conclusion: The findings suggest that PPC promotes angiogenesis by activating the VEGFA/VEGFR2 and downstream signaling pathways, thereby accelerating fracture healing. This study highlights the potential of PPC not only as a liver repair drug but also as a therapeutic agent in enhancing fracture healing through its pro-angiogenic effects.

Liu M, et al. International Journal of Pharmaceutics, 2022, 612, 121365.

PEGylation is known for extending the circulation time of nanocarriers, but it also triggers the accelerated blood clearance (ABC) phenomenon. However, the effect of different PEG chain types on the ABC phenomenon has not been thoroughly explored. This study investigates the influence of linear and branched PEG chain types on the ABC phenomenon by modifying nanoemulsions with 40 kDa molecular weight linear PEG lipid derivatives (DSPE-mPEG40k) and branched PEG lipid derivatives (DSPE-mPEG2,40k).

Preparation of Nanoemulsions

Nanoemulsions were prepared using linear and branched PEG-modified lipids. The oil phase, comprising E80, MCT, and either DSPE-mPEG2,40k, DSPE-mPEG40k, or DSPE-mPEG2k, was constantly stirred in a 55°C water bath. The aqueous phase, also heated to 55°C, was added to the oil phase under rapid stirring. This mixture was stirred for 20 minutes to form nanoemulsions containing phospholipids at a concentration of 10 μmol/mL. The primary nanoemulsions were then ultrasonically treated at 200 W for 3 minutes and at 400 W for 6 minutes using an ultrasonic cell pulverizer. Finally, the nanoemulsions were homogenized by sequentially passing them through polycarbonate membranes with pore sizes of 0.8, 0.45, and 0.22 μm. The resulting nanoemulsions were designated as PE2k (DSPE-mPEG2k), PE40k (DSPE-mPEG40k), and PE2,40k (DSPE-mPEG2,40k).

Results

Characterization of PE40k and PE2,40k nanoemulsions demonstrated favorable physicochemical properties, including size, polydispersity index (PDI), and zeta potential. Pharmacokinetic studies revealed that repeated injections of both PE40k and PE2,40k nanoemulsions did not induce the ABC phenomenon. Notably, PE2,40k exhibited a prolonged circulation time and did not trigger the ABC phenomenon even after repeated injections. This effect is likely due to the lower anti-PEG IgM levels induced by PE2,40k compared to linear PEG-modified nanocarriers, which did not activate the complement system.

The reduced immunogenicity and complement activation of PE2,40k suggest that branched PEG lipid derivatives may offer a more effective solution for extending nanocarrier circulation time without compromising safety and efficacy.