In vivo Targeting of DNA Vaccines to Dendritic Cells via the Mannose Receptor Induces Long-Lasting Immunity against Melanoma.

Herein, we report effective, C-type lectin mannose receptor (MR)-selective, in vivo dendritic cell (DC)-targeting lipid nanoparticles (LNPs) of a novel lipid-containing mannose-mimicking di-shikimoyl- and guanidine head group and two n-hexadecyl hydrophobic tails (DSG). Subcutaneous administration of LNPs of the DSG/p-CMV-GFP complex showed a significant expression of green fluorescence protein in the CD11c+ DCs of the neighboring lymph nodes compared to the control LNPs of the BBG/p-CMV-GFP complex. Mannose receptor-facilitated in vivo DC-targeted vaccination (s.c.) with the electrostatic complex of LNPs of DSG/pCMV-MART1 stimulated long-lasting (270 days post B16F10 tumor challenge) antimelanoma immunity under prophylactic conditions. Remarkably, under therapeutic settings, vaccination (s.c.) with LNPs of the DSG/pCMV-MART1 complex significantly delayed melanoma growth and improved the survival of mice with melanoma. These findings demonstrate that this nonviral delivery system offers a resilient and potential approach to deliver DNA vaccines encoding tumor antigens to DCs in vivo with high efficacy.

Genetic Immunization With In Vivo Dendritic Cell-targeting Liposomal DNA Vaccine Carrier Induces Long-lasting Antitumor Immune Response.

A major limiting factor retarding the clinical success of dendritic cell (DC)-based genetic immunizations (DNA vaccination) is the scarcity of biologically safe and effective carrier systems for targeting the antigen-encoded DNA vaccines to DCs under in vivo settings. Herein, we report on a potent, mannose receptor selective in vivo DC-targeting liposomes of a novel cationic amphiphile with mannose-mimicking shikimoyl head-group. Flow cytometric experiments with cells isolated from draining lymph nodes of mice s.c. immunized with lipoplexes of pGFP plasmid (model DNA vaccine) using anti-CD11c antibody-labeled magnetic beads revealed in vivo DC-targeting properties of the presently described liposomal DNA vaccine carrier. Importantly, s.c. immunizations of mice with electrostatic complex of the in vivo DC-targeting liposome and melanoma antigen-encoded DNA vaccine (p-CMV-MART1) induced long-lasting antimelanoma immune response (100 days post melanoma tumor challenge) with remarkable memory response (more than 6 months after the second tumor challenge). The presently described direct in vivo DC-targeting liposomal DNA vaccine carrier is expected to find future exploitations toward designing effective vaccines for various infectious diseases and cancers.

Novel Suberoylanilide Hydroxamic Acid Analogs Inhibit Angiogenesis and Induce Apoptosis in Breast Cancer Cells.

BACKGROUND: Histone deacetylases (HDACs) are the enzymes that catalyze the removal of the acetyl group from lysine residues and regulate several biological processes. Suberoylanilide hydroxamic acid (SAHA) is a notable HDAC inhibitor that exhibited remarkable anti-proliferative efficiency by alleviating gene regulation against solid and hematologic cancers. AIM: The aim of this study was to develop new chemotherapeutic agents for breast cancer treatment, therefore, a novel series of Suberoylanilide hydroxamic acid (SAHA) analogs were investigated as anticancer agents. METHODS: We designed and synthesized a novel series of analogs derived from SAHA by substituting alkyl, alkoxy, halo, and benzyl groups at different positions of the phenyl ring. The newly synthesized analogs were assessed for their cytotoxic potential against four human cancer cell lines in comparison with healthy cell lines, using several biological assays. RESULTS: SAHA analogs displayed significant cytotoxic potential with IC50 values ranging from 1.6 to 19.2 µM in various tumor cell lines. Among these analogs, 2d (containing 3-chloro, 4-floro substitutions on phenyl moiety), 2h (containing 3,4-di chloro substitutions on phenyl moiety), and 2j (containing 4-chloro, 3-methyl substitutions on phenyl moiety) showed significant cytotoxic potential with IC50 values ranging from 1.6 to 1.8 µM in MCF-7 (breast carcinoma) cell line. More importantly, these analogs were found to be non-toxic towards healthy primary human hepatocytes (PHH) and mouse fibroblast cells (NIH3T3), which represent their tumor selectivity. These analogs were further analyzed for their effect on cell migration, BrdU incorporation, Annexin V-FITC and cell cycle arrest (Sub-G1 phase). Remarkably, analogs 2d, 2h, and 2j displayed significant HDAC inhibition than the parent SAHA molecule. Further studies also confirmed that these SAHA analogs are efficient in inducing apoptosis, as they regulated the expression of several proteins involved in mitochondrial or intrinsic apoptosis pathways. Findings in the Chick Chorioallantoic Membrane (CAM) assay studies revealed anti-angiogenic properties of the currently described SAHA analogs. CONCLUSION: From anti-proliferative study results, it is clearly evident that 3,4-substitution at the SAHA phenyl ring improves the anti-proliferative activity of SAHA. Based on these findings, we presume that the synthesized novel SAHA analogs could be potential therapeutic agents in treating breast cancer.

Amphetamine decorated cationic lipid nanoparticles cross the blood-brain barrier: therapeutic promise for combating glioblastoma.

Combating brain tumors (glioblastoma multiforme or GBM) is a formidable challenge because of the existence of blood-brain barrier (BBB), a tight cellular junction that separates the central nervous system (CNS) and systemic circulation. Such a selectively permeable barrier prevents the entry of therapeutic molecules from blood circulation to brain parenchyma. Towards enhancing the efficacy of brain tumor-selective drug delivery without perturbing the BBB integrity, nanometric drug carriers are increasingly becoming an efficient therapeutic modality in preclinical studies. Psychostimulant drugs such as amphetamine and methylated amphetamine (METH) are known to penetrate the BBB. Still, little effort has been made to exploit them in nano-drug delivery, largely due to their toxicities. Herein, for the first time, we design, synthesize, and formulate three different ß-amphetaminylated cationic lipid nanoparticles. We show that the ß-amphetaminylated cationic lipid nanoparticles are nontoxic and can cross the BBB presumably through active transcytosis. The BBB penetrating ability also depends on the hydrophilic-hydrophobic balance of the lipids, with hexadecyl lipid (16-BACL) nanoparticle showing maximum accumulation in the brain. The lipid nanoparticle of 16-BACL can simultaneously encapsulate paclitaxel and PDL1-siRNA. The dual drug-loaded lipid nanoparticles showed apoptosis driven cellular cytotoxicity against GL261 cells and improved the overall survivability of orthotopic glioblastoma bearing mice compared to their non-targeting counterpart. The present work describes a new class of BBB-crossing lipid nanoparticles and delineates their therapeutic promise against glioblastoma.

Improving Payload Capacity and Anti-Tumor Efficacy of Mesenchymal Stem Cells Using TAT Peptide Functionalized Polymeric Nanoparticles.

Mesenchymal stem cells (MSCs) accumulate specifically in both primary tumors and metastases following systemic administration. However, the poor payload capacity of MSCs limits their use in small molecule drug delivery. To improve drug payload in MSCs, we explored polymeric nanoparticles that were functionalized with transactivator of transcription (TAT) peptide. Paclitaxel loaded poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (15⁻16% w/w paclitaxel; diameter of 225 ± 7 nm; and zeta potential of -15 ± 4 mV) were fabricated by emulsion-solvent evaporation method, followed by TAT-conjugation to the surface of nanoparticles via maleimide-thiol chemistry. Our studies demonstrated that TAT functionalization improved the intracellular accumulation and retention of nanoparticles in MSCs. Further, nano-engineering of MSCs did not alter the migration and differentiation potential of MSCs. Treatment with nano-engineered MSCs resulted in significant (p < 0.05) inhibition of tumor growth and improved survival (p < 0.0001) in a mouse orthotopic model of lung cancer compared to that with free or nanoparticle encapsulated drug. In summary, our results demonstrated that MSCs engineered using TAT functionalized nanoparticles serve as an efficient carrier for tumor specific delivery of anticancer drugs, resulting in greatly improved therapeutic efficacy.

In vivo targeting of DNA vaccines to dendritic cells using functionalized gold nanoparticles.

The clinical success of dendritic cell (DC)-based genetic immunization remains critically dependent on the availability of effective and safe nano-carriers for targeting antigen-encoded DNA vaccines to DCs, the most potent antigen-presenting cells in the human body in vivo. Recent studies revealed the efficacies of mannose receptor-mediated in vivo DC-targeted genetic immunization by liposomal DNA vaccine carriers containing both mannose-mimicking shikimoyl and transfection enhancing guanidinyl functionalities. However, to date, the efficacies of this approach have not been examined for metal-based nanoparticle DNA vaccine carriers. Herein, we report for the first time, the design, synthesis, physico-chemical characterization and bioactivities of gold nanoparticles covalently functionalized with a thiol ligand containing both shikimoyl and guanidinyl functionalities (Au-SGSH). We show that Au-SGSH nanoparticles can deliver DNA vaccines to mouse DCs under in vivo conditions. Subcutaneous administration of near infrared (NIR) dye-labeled Au-SGSH showed significant accumulation of the NIR dye in the DCs of the nearby lymph nodes compared to that for the non-targeting NIR-labeled Au-GSH nanoconjugate containing only a covalently tethered guanidinyl group, not the shikimoyl-functionality. Under prophylactic settings, in vivo immunization (s.c.) with the Au-SGSH-pCMV-MART1 nanoplex induced a long-lasting (180 days) immune response against murine melanoma. Notably, mannose receptor-mediated in vivo DC-targeted immunization (s.c.) with the Au-SGSH-MART1 nanoplex significantly inhibited established melanoma growth and increased the overall survivability of melanoma-bearing mice under therapeutic settings. The Au-SGSH nanoparticles reported herein have potential use for in vivo DC-targeted genetic immunization against cancer and infectious diseases.

Delivering anti-cancer drugs with endosomal pH-sensitive anti-cancer liposomes.

Numerous prior studies have been reported on the use of pH-sensitive drug carriers such as micelles, liposomes, peptides, polymers, nanoparticles, etc. that are sensitive to the acidic (pH = ∼6.5) microenvironments of tumor tissues. Such systems have been primarily used in the past as effective drug/gene/microRNA carriers for releasing their anti-cancer payloads selectively to tumor cells/tissues. Herein, we report on the development of new liposomal drug carriers prepared from glutamic acid backbone-based cationic amphiphiles containing both endosomal pH-sensitive histidine as well as cellular uptake & solubility enhancing guanidine moieties in their polar head-group regions. The most efficient one among the four presently described endosomal pH-sensitive liposomal drug carriers not only effectively delivers potent anti-cancer drugs (curcumin & paclitaxel) to mouse tumor, but also significantly contributes to inhibiting mouse tumor growth. The findings in the in vitro mechanistic studies are consistent with apoptosis of tumor cells being mediated through increased cell cycle arrest in the G2/M phase. Findings in the FRET assay and in vitro drug release studies conducted with the liposomes of the most efficient pH-sensitive lipid demonstrated its pH dependent fusogenic and controlled curcumin release properties. Importantly, the presently described liposomal formulation of curcumin & paclitaxel enhanced overall survivability of tumor bearing mice. To the best of our knowledge, the presently described system (curcumin, paclitaxel and liposomal carrier itself) is the first of its kind pH-sensitive liposomal formulation of potent chemotherapeutics in which the liposomal drug itself exhibits significant mouse tumor growth inhibition properties.

Examples of Tumor Growth Inhibition Properties of Liposomal Formulations of pH-Sensitive Histidinylated Cationic Amphiphiles.

Herein we report on the unexpected cancer cell selective cytotoxicities of the liposomal formulations of aspartic and glutamic acid backbone-based four novel lipids with endosomal pH-sensitive head-groups and aliphatic n-hexadecyl & n-octadecyl hydrophobic tails. Surprisingly, although the formulations killed cancer cells efficiently, they were significantly less cytotoxic in non-cancerous healthy cells. Importantly, intratumoral administration of the liposomal formulations efficiently inhibited growth of melanoma in a syngeneic C57BL/6J mouse tumor model. Western Blotting experiments with the lysates of liposomes treated cancer cells revealed that liposomes of lipids 1-4 induce apoptosis selectively in cancer cells presumably by releasing cytochrome c from depolarized mitochondria and subsequent activation of caspases 3 & 9, upregulation of Bax and down regulation of Bcl-2. In summary, the present report describes for the first time tumor growth inhibition properties of the liposomal formulations of endosomal pH-sensitive histidinylated cationic lipids under both in vitro and systemic settings.

A long-lasting dendritic cell DNA vaccination system using lysinylated amphiphiles with mannose-mimicking head-groups.

Dendritic cells (DCs) pulsed/transduced with tumor-associated or viral antigens have shown promise in combating cancer and infectious diseases. Despite significant progresses, development of a biologically safe DC-based genetic immunization (DNA vaccination) system capable of providing truly long-lasting protective immunity remains a significant scientific challenge. Here we show that immunization with autologous DCs pre-transfected with electrostatic complexes (lipoplexes) of a plasmid DNA encoding melanoma tumor associated antigen and liposomes of two lysinylated cationic amphiphiles with mannose-mimicking quinic and shikimic acid head-groups provides long-lasting (300 days post tumor challenge) protective immunity with significant memory response (more than six months after the second tumor challenge) in more than 80% immunized mice. The presently described non-viral ex vivo DC-transfection system may be exploited in inducing long-lasting immune response in DC-based genetic immunization.