Hepatic Carcinoma Selective Nucleic Acid Nanovector Assembled by Endogenous Molecules Based on Modular Strategy.

We rationally formulated a nucleic acid nanovector platform utilizing endogenous molecules in the following steps: nucleic acids are initially packed by a multifunctional peptide and a cationic liposome to form positively charged ternary complexes through electrostatic interaction; then the ternary complexes were coated with hyaluronic acid (HA) to form negatively charged quaternary nanocomplexes (Q-complexes). Among the components of Q-complexes, the multifunctional peptide was composed of a poly-16-arginine (R16) and a hepatic tumor-targeted cell penetrating peptide (KRPTMRFRYTWNPMK); the cationic lipid component included DOTAP and fusogenic lipid DOPE; the HA component shielded the cationic ternary complexes and actively targeted the CD44 overexpressed on the surface of tumor cells. Q-complexes have showed a relatively high stability in the medium, and HA component partially separated from the nanocomplexes after the Q-complexes bound to the cancer cells. The Q-complexes showed significantly enhanced nucleic acid delivery activity than the corresponding quaternary complexes containing R16 and nonvisible cytotoxicity in SCMM-7721 cells. In vivo, a selected Q-complex HLP1R specifically targeted and entered tumor cells without affecting normal tissues. Furthermore, HLP1R wrapped survivin siRNA efficiently and silenced the targeting gene in the liver orthotropic transplantation tumor models and showed nontoxic in vivo. This study reveals that Q-complexes are reasonable and feasible gene therapeutic carriers.

Liposomal T cell engager and re-director for tumor cell eradication in cancer immunotherapy.

T cells are one of the most important effector cells in cancer immunotherapy. Various T cell-dependent bispecific antibody (TDB) drugs that engage T cells for targeted cancer cell lysis are being developed. Here, we describe supra-molecular T-cell redirecting antibody fragment-anchored liposomes (TRAFsomes) and report their immune modulation and anti-cancer effects. We found that TRAFsomes containing different copies of anti-CD3 fragments displayed different T cell modulation profiles, showing that optimization of surface density is needed to define the therapeutic window for potentiating cancer cell-specific immune reactions while minimizing nonspecific side effects. Moreover, small molecular immunomodulators may also be incorporated by liposomal encapsulation to drive CD8 + T cell biased immune responses. In vivo studies using human peripheral blood mononuclear cell reconstituted mouse models showed that TRAFsomes remained bounded to human T cells and persisted for more than 48 hours after injection. However, only TRAFsomes containing a few anti-CD3 (n = 9) demonstrated significant T cell-mediated anti-cancer activities to reverse tumor growth. Those with more anti-CD3s (n = 70) caused tumor growth and depletion of human T cells at the end of treatments. These data suggested that TRAFsomes can be as potent as traditional TDBs and the liposomal structure offers great potential for immunomodulation and improvement of the therapeutic index.Abbreviation: Chimeric antigen receptor T cells (CAR-T cells), Cytokine release syndrome (CRS) Cytotoxic T cell (CTL) Effector: target ratios (E:T ratios), Heavy chain (HC) Immune-related adverse events (irAE), Large unilamellar vesicle (LUV), Peripheral blood mononuclear cells (PBMCs, Single-chain variable fragment (scFv), T cell-dependent bispecific antibody (TDB), T cell redirecting antibody fragment-anchored liposomes (TRAFsomes), Methoxy poly-(ethylene glycol) (mPEG).

Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design.

A safe and efficient quaternary gene delivery system (named Q-complexes) was constructed based on self-assembly of molecules through noncovalent bonds. This system was formulated through the cooperation and competing interactions of cationic liposomes, multifunctional peptides, and DNA, followed by coating hyaluronic acid on the surface of the ternary complexes. The multifunctional peptide was composed of two functional domains: penetrating hepatic tumor-targeted cell moiety (KRPTMRFRYTWNPMK) and a wrapping gene sequence (polyarginine 16). The effect of spacer insertion between the two domains of multifunctional peptide on the intracellular transfection of Q-complexes was further studied. Experimental results showed that the formulations assembled with various peptides in the spacer elements possessed different intercellular pathways and transfection efficiencies. The Q-complexes containing peptide in the absence of spacer element (Pa) showed the highest gene expression among all samples. The Q-complexes containing peptides with a noncleavable spacer GA (Pc) had no ability of intracellular nucleic acid delivery, whereas those with a cleavable spacer RVRR (Pd) showed moderate transfection activity. These results demonstrated that the different spacers inserted in the multifunctional peptide played an important role in in vitro DNA transfection efficiency. Atomic force microscopy images showed that the morphologies of ternary complexes (LPcD) and Q-complexes (HLcPD) were crystal lamellas, whereas those of other nanocomplexes were spheres. Circular dichroism showed the changed configuration of peptide with spacer GA in nanocomplexes compared with that of its free state, whereas the Pa configuration without spacer in nanocomplexes was consistent with that of its free state. The present study contributed to the structural understanding of Q-complexes, and further effective modification is in progress.