Recent advancements in liposome technology

Purpose: Liposomes remain an important nano-sized drug delivery system because of their biomimetic bilayer, ease of preparation, and biocompatibility, but clinical translation is still limited by reproducibility, scale-up, stability, and payload-specific barriers. The review updates recent advances in liposome preparation, excipients, administration routes, and novel modalities including nucleic acid therapies, CRISPR/Cas9, and immunotherapies.

Hypothesis: As a review, there is no single experimental hypothesis. Central thesis: if liposome production techniques, excipient design, analytical characterization, administration routes, and scale-up strategies continue to advance, then liposomal delivery can be broadened to nucleic acid therapeutics, CRISPR/Cas9 genome editing, gas delivery, and cancer immunotherapy, improving clinical translation.

Aims: Review recent liposome preparation techniques, especially microfluidic methods. - Summarize excipients used in novel liposomal formulations. - Discuss routes of administration for targeted disease delivery, including oral, transdermal, and blood–brain barrier crossing. - Highlight payload classes: siRNA, microRNA, mRNA, pDNA, CRISPR/Cas9, gases, and immunotherapeutics. - Cover analytical development for size/zeta potential and plasma drug release. - Address scaling up of liposome production and future nanotechnological approaches.

Delivery system: Platform: Liposomes and modified liposomes, including conventional, PEGylated/stealth, cationic, anionic, neutral, elastic/transferosomes, bubble liposomes, porphyrin-phospholipid liposomes, magnetoliposomes, and Trojan horse liposomes. - Payloads: siRNA, microRNA, mRNA, pDNA, CRISPR/Cas9 (Cas9 protein/sgRNA, Cas9 mRNA/sgRNA, or plasmid), gases such as nitric oxide and carbon monoxide, immunotherapeutics such as cGAMP, antigens, adjuvants, and conventional small-molecule drugs. - Key lipids: Cationic lipids DOTAP, DODAP, DODMA, DOTMA, DC-Chol, DLin-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DLin-K-DMA; helper lipids DOPE, cholesterol, DSPC, DOPC; PEGylated lipids. - Targeting/functionalization: transferrin, RGD, R8-dGR, glucose, glutathione, antibodies, peptides, pH-sensitive, fusogenic, and temperature-sensitive moieties.

Approach: Review of recent literature and clinical trial landscape. No new primary experimental data. Covers in vitro cell lines such as HEK293, A549, SK-OV-3, C26, BHT-101, U87, and others; in vivo models including mice, rats, canine, and macaque; disease contexts include cancer, cystic fibrosis, osteoporosis, mucopolysaccharidosis I, type II diabetes, hindlimb ischemia, Alzheimer’s disease, Parkinson’s disease, and glioma. Clinical trials are summarized across Phase I–III.

Key methods: Microfluidic preparation methods: flow focusing, pulsed jetting, double emulsion templating, droplet emulsion transfer, hydrodynamic focusing, transient membrane ejection, ice droplet hydration, staggered herringbone micromixer. - Characterization: size and zeta potential by light scattering, cryo-TEM, freeze-fracture TEM, field flow fractionation, nanoparticle tracking, etc. - Plasma drug release quantification: solid-phase extraction (SPE) with LC-MS/MS, ultrafiltration, ion-exchange chromatography, size-exclusion chromatography, capillary electrophoresis. - Functional assays: gene editing efficiency, miRNA downregulation, VEGF-A expression, tumor growth inhibition, survival, BBB penetration, and toxicity.

Key results: DOTAP-based liposome delivery of Cas9 and sgRNA plasmid achieved 39% gene-editing efficiency in knocking out a GFP reporter in HEK293 cells. - DOTAP/Chol/DOPE liposomes delivering miR-34b-5p to thyroid carcinoma reduced VEGF-A expression 2.5-fold and reduced tumor size in a BHT-101 xenograft. - Liposomal antagomiR-148a produced ~80% downregulation of miR-148a in osteoporotic mice, reducing bone resorption without liver or kidney toxicity. - A multifunctional liposome containing epirubicin, tamoxifen, and transferrin achieved 52.2% cytostatic penetration across the BBB vs free epirubicin, and median survival increased to 23 days vs 15 days for free epirubicin in a mouse model. - SPE-LC-MS/MS separation achieved LLOQ of 3.13 ng/mL for non-liposomal doxorubicin and 0.156 µg/mL for liposomal doxorubicin; for amphotericin B, LLOQ was 0.2 µg/mL free and 0.5 µg/mL liposomal. - Microfluidic flow focusing produced monodisperse liposomes around 50–150 nm; some emulsion-transfer vesicles remained stable for >26 days.

Interpretation: Liposomes are versatile drug delivery systems with feasible excipient compositions and chemical modifications, and they have been administered safely by multiple routes. Since their discovery, production technology has advanced through new lipid components and preparation procedures, especially microfluidics. Although many FDA-approved liposomal therapeutics exist and more are in development, clinical needs are not yet fully met. Recent progress in encapsulation efficiency, reproducibility, and size control justifies continued development of liposomal nanomedicine.

Limitations: This is a review, not a primary experimental study. Key hurdles include batch-to-batch reproducibility, low entrapment for some drugs, effective sterilization, shelf stability, and scale-up for clinical use. Multifunctional liposome production remains challenging and costly. PEGylated liposomes can generate anti-PEG antibodies. CRISPR/Cas9 delivery faces RES clearance, immune responses, and insufficient target tissue accumulation. Oral liposome absorption mechanisms are poorly understood; transdermal liposomes face storage and skin-stability challenges; BBB delivery remains difficult. Many novel applications remain preclinical.