Cationic liposomes as gene delivery system: transfection efficiency and new application

Purpose: Cationic liposomes (CLs) are attractive non-viral gene-delivery carriers because they are safer, less immunogenic, and easier to prepare than viral vectors, but their transfection efficiency (TE) remains low and the relationship between lipoplex physicochemical properties, lipoplex–cell interaction, and TE is poorly understood. This review describes recent work on CL–DNA complex properties and factors influencing TE, and introduces recent therapeutic applications.

Hypothesis: As a review, there is no single experimental hypothesis. Central thesis: if the physicochemical properties of cationic liposome–gene complexes—lipid structure, PEGylation, lipid molar ratio, (+/−) charge ratio, vesicle size, and medium conditions—are understood and optimized, then CL-mediated transfection efficiency can be improved and CLs can be applied more effectively in gene therapy.

Aims: Introduce CLs and their materials, structures, and transfection mechanisms. - Describe recent work elucidating the relationship between the chemical–physical properties of CL–DNA complexes and TE in mammalian cells. - Review factors influencing TE: lipid structure, PEGylation, lipid molar ratio and (+/−) charge ratio, vesicle size, and medium conditions. - Summarize recent applications of CLs in gene therapy, including cancer, bone damage, infection, and co-delivery of gene and drug.

Delivery system: Platform: Cationic liposomes (CLs) and lipoplexes. - Cationic lipids: DOTMA, DOTAP, DMRIE, DOSPA, DODAG, Arg-Glu2C16, DE, cholesterol-based cationic lipids, OH-Chol, and asymmetric divalent head group cholesterol-based lipids. - Neutral co-lipids: DOPE, cholesterol (Chol), DOPC. - Payloads: plasmid DNA (pDNA), antisense oligonucleotides (ASODN), mRNA, peptide-nucleic acids (PNAs), siRNA; also co-delivery with drugs such as vinblastine and doxorubicin. - Structures: multilamellar \(L_\alpha^C\) phase with DNA monolayers sandwiched between cationic membranes; inverted hexagonal \(H_{II}^C\) phase with DNA encapsulated in inverse cylindrical micelles; “beads-on-a-string” complex. - Surface modification: PEGylation, transferrin, truncated human basic fibroblast growth factor peptide, etc.

Approach: Review/synthesis of in vitro and in vivo preclinical and clinical studies. In vitro cell lines include K562 erythroleukemia, 5637 epithelial carcinoma, neuronal cells, TSA mammary adenocarcinoma, and others. In vivo examples include B16F10 mouse melanoma, tuberculosis vaccine models, transcutaneous immunization against Japanese encephalitis virus, and osteoblast-like cell gene transfer. Not a systematic review.

Key methods: X-ray structural studies, statistical mechanical models, fluorescence microscopy, confocal laser scanning microscopy (CLSM), antigen inhibition effect assay, β-galactosidase expression scoring, transfection efficiency assays, and in vitro/in vivo gene expression and antitumor activity measurements.

Key results: CL–DNA complexes exhibit structural polymorphism: lamellar \(L_\alpha^C\) and inverted hexagonal \(H_{II}^C\) phases; DOPE tends to adopt the \(H_{II}\) phase and may aid endosomolysis, while cholesterol changes interlamellar spacing. - Amino acid-based CL Arg-Glu2C16 showed 4-fold higher TE than Lipofectamine 2000 in neuronal cells. - Of 24 asymmetric divalent head group cholesterol-based cationic lipids, 7 exhibited higher transfection efficiency than commercial agents; compound 5 had the highest TE. - DC-Chol/DOPE liposomes were most efficient for pDNA or siRNA at 1:2 or 1:1 molar ratio, respectively. siRNA TE was positively associated with DC-Chol/siRNA weight ratio, whereas pDNA TE decreased as DC-Chol/pDNA weight ratio increased. - PEGylation lowered cellular interaction and uptake, and decreased pDNA/siRNA TE of DC-Chol/DOPE CLs and DOTAP/DOPE CLs carrying oligonucleotides. - Vesicle size effects remain disputed: in K562 cells, unilamellar GLB73 (~120 nm) gave highest lipofection, followed by MLVs GLB391 (~650 nm) and unilamellar (~100 nm); MLVs GLB43 (~270 nm) were more effective than unilamellar (~120 nm). Some studies associate higher activity with smaller complexes (~200 nm or less), while others report larger MLVs (~900 nm) outperform SUVs (~25 nm). - In application, simultaneous vinblastine and HSA-EPOPC:Chol/DNA (+/−) (4/1) lipoplexes improved transgene expression more than 10 times and enhanced antitumoral activity in TSA cells.

Interpretation: CL–gene complexes have unique structures and properties whose interactions strongly affect transfection behavior. To develop more efficient CL gene carriers, the complex and sometimes contradictory results—especially regarding particle diameter, serum effects, and lipoplex structure—must be clarified. CLs are less toxic, less immunogenic, and easier to prepare than viral vectors, making them potentially attractive for clinical applications; co-delivery of gene and drug is a promising direction.

Limitations: This is a review, not a primary experimental study. TE remains low compared to viral vectors. The literature contains conflicting results on key factors such as vesicle size and PEGylation. Serum substantially lowers TE in most cases. Clinical translation is not established, and the review is not comprehensive or systematic.