Liposome-Coated Lipoplex-Based Carrier for Antisense Oligonucleotides

Purpose: Most cancer gene therapy research has focused on solid tumors, while blood cancers such as leukemia and lymphoma have received less attention. There is a need for a lipid carrier that protects antisense oligonucleotides or plasmid DNA from enzymatic degradation, is stable in serum, has low toxicity, and can efficiently deliver cargo to leukemia/lymphoma cells and inhibit tumor growth in vivo.

Hypothesis: If a liposome-coated lipoplex (L-cL) is constructed with a cationic DOTAP–nucleic acid core and a PEG-containing lipid bilayer coat, then it will protect encapsulated antisense oligodeoxynucleotides (asODNs) or plasmid DNA, transfect leukemia/lymphoma cells efficiently, silence BCL-2 expression, reduce cell survival, prolong circulation, and inhibit tumor growth in vivo.

Aims: Develop an original lipid composition for a liposome-coated lipoplex carrier for nucleic acids. - Characterize the carrier for size, zeta potential, encapsulation efficiency, storage stability, serum stability, and protection against DNase/serum nucleases. - Evaluate transfection efficiency in leukemia/lymphoma cell lines using a GFP-encoding plasmid. - Test BCL-2-directed asODN delivery for mRNA/protein silencing and effects on cell survival in vitro. - Assess biodistribution, blood circulation, and antitumor efficacy in mice.

Delivery system: Platform: Liposome-coated lipoplex (L-cL). - Core: DOTAP complexed with asODN or plasmid DNA at a DOTAP:nucleic acid ratio of 1:20 (w/w). - Coating bilayer: HPC, DC-CHOL, DOPE, and DSPE-PEG at a 0.5:0.2:0.1:0.2 (w/w) ratio. - Core DOTAP to bilayer HPC ratio: 0.7. - Payloads: BCL-2-directed asODN, scrambled ODN, or pEGFP plasmid DNA. - Targeting ligand: None; the carrier is PEG-modified but not actively targeted. - Physicochemical properties: Diameter 116.3 ± 7.8 nm; zeta potential 6.6 ± 0.9 mV; encapsulated pDNA ~120 µg/ml, with 80–95% entrapment.

Approach: In vitro cell lines: Jurkat T, HL-60, Daudi, K562, and primary AML patient white blood cells. - In vitro conditions: Transfection assessed in PBS, serum-free medium, or medium supplemented with 10% FBS; incubation for 24–48 h depending on assay. - In vivo biodistribution: BALB/c mice injected via tail vein with DiD-labeled L-cL or control PE/PC liposomes. - In vivo efficacy: Daudi Burkitt’s lymphoma cells implanted subcutaneously into NOD/SCID mice; 60 males total. Treatments were PBS, L-cL containing BCL-2 asODN, or L-cL containing scrambled ODN. - Dosing: 50 mg/kg liposomes containing 0.8 mg ODN/kg, administered intravenously weekly on days 7, 14, 21, and 28. - Controls: Naked asODN, scrambled ODN in L-cL, PBS, and non-PEGylated PE/PC liposomes.

Key methods: Dynamic light scattering / Zetasizer for size and zeta potential. - Spectrophotometric absorbance at 260 nm for encapsulated nucleic acid content. - Agarose gel electrophoresis for DNase I and human serum protection. - Fluorescence microscopy for GFP transfection efficiency. - RT-PCR and Western blot for BCL-2 mRNA and protein silencing. - Trypan Blue exclusion and MTT assays for cell viability/toxicity. - Hemolysis assay with human erythrocytes. - In vivo fluorescence imaging (NightOwl II LB 983) and spectrofluorimetry for biodistribution/blood circulation. - Tumor volume measurement for therapeutic efficacy; TGI calculated.

Key results: L-cL size was 116.3 ± 7.8 nm; zeta potential 6.6 ± 0.9 mV. - Encapsulation efficiency for pDNA was 80–95%; pDNA content ~120 µg/ml. - Stable as a suspension for 12 months at 4°C; stable after freeze-drying for 3 months at −20°C; stable in 50% human serum for up to 30 h. - Transfection efficiency in HL-60 and Jurkat T cells reached ~80–85% with serum-containing medium. At 2.5 µg/ml pDNA: HL60 82 ± 2% and Jurkat T 80 ± 3% at 24 h; HL60 83 ± 4% and Jurkat T 79 ± 2% at 48 h. - BCL-2 asODN in L-cL silenced BCL-2 mRNA and protein; naked asODN and scrambled ODN showed no effect. - After 24 h with 2.5 µg/ml BCL-2 asODN, survival was ~40% for Jurkat T, ~50% for HL60, and ~59% for Daudi cells. - In primary AML patient cells, 40–50% survived at 24–48 h and ~25% at 72 h. - In vivo, PEG-modified L-cL remained detectable >8 h and in blood up to 24 h; >60% maximal blood concentration up to 12 h; ~25% fluorescence still detectable at 24 h. Control PE/PC liposomes were essentially undetectable by 12 h. - Tumor growth inhibition (TGI) for L-cL asODN was 60.38% on day 21, remained 55–60%, and was 57% on day 30. Scrambled ODN L-cL gave 0.33% TGI on day 30. - Toxicity: hemolysis ≤5%; neutral pDNA-containing L-cL showed minimal toxicity, with cell survival generally >80% at tested concentrations; highest doses showed some toxicity. Body weight loss in treated mice was <5%.

Interpretation: The authors conclude that L-cL is a stable, low-toxicity, nonviral lipid carrier that protects nucleic acids, achieves high transfection in difficult-to-transfect leukemia/lymphoma cells, effectively silences BCL-2, prolongs circulation, and significantly inhibits tumor growth in a Daudi lymphoma xenograft model. They propose it as a promising carrier for antisense therapy in blood cancers, potentially useful in combination with cytostatic agents.

Limitations: No active targeting ligand; delivery relies on passive/PEG-modified liposomal behavior. - Biodistribution showed substantial accumulation in liver and spleen, with low signal in lung, kidney, and brain. - In vitro BCL-2 silencing and AML patient cell data are limited; primary AML data came from a single patient sample. - In vivo efficacy was tested only in a Daudi xenograft in immunodeficient mice, with no large-animal validation. - Therapeutic effect was assessed as monotherapy; combination with cytostatics was suggested but not tested. - Long-term storage showed up to ~25% change in ODN content over 6 months.