Newly Engineered Magnetic Erythrocytes for Sustained and Targeted Delivery of Anti-Cancer Therapeutic Compounds

Cytotoxic chemotherapy of cancer is limited by serious, sometimes life-threatening, side effects that arise from toxicities to sensitive normal cells because the therapies are not selective for malignant cells. So how can they be selectively improved? Alternative pharmaceutical formulations of anti-cancer agents have been investigated in order to improve conventional chemotherapy treatment. These formulations are associated with problems like severe toxic side effects on healthy organs, drug resistance and limited access of the drug to the tumor sites suggested the need to focus on site-specific controlled drug delivery systems. In response to these concerns, we have developed a new drug delivery system based on magnetic erythrocytes engineered with a viral spike fusion protein. This new erythrocyte-based drug delivery system has the potential for magnetic-controlled site-specific localization and highly efficient fusion capability with the targeted cells. Here we show that the erythro-magneto-HA virosomes drug delivery system is able to attach and fuse with the target cells and to efficiently release therapeutic compounds inside the cells. The efficacy of the anti-cancer drug employed is increased and the dose required is 10 time less than that needed with conventional therapy

Novel epigenetic target therapy for prostate cancer: a preclinical study.

Epigenetic events are critical contributors to the pathogenesis of cancer, and targeting epigenetic mechanisms represents a novel strategy in anticancer therapy. Classic demethylating agents, such as 5-Aza-29-deoxycytidine (Decitabine), hold the potential for reprograming somatic cancer cells demonstrating high therapeutic efficacy in haematological malignancies. On the other hand, epigenetic treatment of solid tumours often gives rise to undesired cytotoxic side effects. Appropriate delivery systems able to enrich Decitabine at the site of action and improve its bioavailability would reduce the incidence of toxicity on healthy tissues. In this work we provide preclinical evidences of a safe, versatile and efficient targeted epigenetic therapy to treat hormone sensitive (LNCap) and hormone refractory (DU145) prostate cancers. A novel Decitabine formulation, based on the use of engineered erythrocyte (Erythro-Magneto-Hemagglutinin Virosomes, EMHVs) drug delivery system (DDS) carrying this drug, has been refined. Inside the EMHVs, the drug was shielded from the environment and phosphorylated in its active form. The novel magnetic EMHV DDS, endowed with fusogenic protein, improved the stability of the carried drug and exhibited a high efficiency in confining its delivery at the site of action in vivo by applying an external static magnetic field. Here we show that Decitabine loaded into EMHVs induces a significant tumour mass reduction in prostate cancer xenograft models at a concentration, which is seven hundred times lower than the therapeutic dose, suggesting an improved pharmacokinetics/pharmacodynamics of drug. These results are relevant for and discussed in light of developing personalised autologous therapies and innovative clinical approach for the treatment of solid tumours

Magnetically Driven Bioreactors as new Tools in Drug Delivery

The pharmacological properties of many drugs can be improved by drug delivery systems able to drive therapeutic agents to target regions. The use of carriers, in fact, may reduce possible cytotoxic effects of drugs and increase their bioavailability at the site of action, thus improving the efficacy and the safety of treatments. Therefore, we have developed an erythrocyte-based drug delivery system (erythro-magneto-HA virosome), which has the potential to be magnetically guided to specific sites and to fuse with target cells. These engineered erythrocytes have demonstrated in previous work a very high in vitro capability to release anticancer drugs directly inside target cells. Because the erythro-magneto-HA virosomes (EMHVs) proved to be promising carriers, we decided to investigate in more details the effectiveness and safety of this erythrocyte-based drug delivery system. We evaluated the ability of the EMHVs to be specifically localized in vivo to desired sites by means of an external magnetic field and to protect an anticancer drug such as 5-Aza-2\u27-deoxycytidine from degradation. Additionally we have assessed the ability of the EMHVs to act as bioreactors and to convert the pro-drug 5-Aza-2\u27-deoxycytidine into an active drug. Finally, we have studied the interaction of the EMHVs with the host immune system. The pro-drug 5-Aza-2\u27-deoxycytidine has short half-life when systemically injected and needs to be phosphorylated to become an active drug. We found that when inside the engineered erythrocytes it is protected by degradation and is transformed in its active form thus becoming readily available for uptake by the targeted cells. Moreover, we have observed that the EMHVs used didn\u27t cause either a cell-mediated or a humoral immune response in host mice having the same haplotype of the donors. These findings suggest that erythro-magneto-HA virosomes are a safe and useful drug delivery system that may offer numerous advantages for several clinical application

HPLC/QTOF quantitative analysis of 5-Aza-dC.

<p>(<b>A</b>) Peaks of 1,14 ng 5-Aza-dC (standard). (<b>B</b>) Peaks of 5-Aza-dC encapsulated into 2×10⁹ erythro-magneto-HA virosomes. <b>RT</b>: retention time; <b>AA</b>: peak area counts. (<b>C</b>) Calibration curve of 5-Aza-dC. The peak area values of standards were put in relation with the ng/10 µl of 5-Aza-2-dC sample ran. Standard solutions used were 1,14, 2,28, 4,56 and 11,40 ng 5-Aza-dC (black rhombuses). Grey triangles show the 5-Aza-dC concentrations found within the two erythro-magneto-HA virosomes samples used in <i>in vitro</i> experiments.</p

Kinetic Fusion assay.

<p>The 5-Aza-2-dC-loaded erythro-magneto-HA virosomes were labelled with Octadecyl Rhodamine (R18) and incubated with HeLa cells. Fusion has been reported as % of R18 fluorescence dequenching (FDQ) using 560 and 590 nm as excitation and emission wavelengths respectively. RBC: 5-Aza-2-dC-loaded magnetic erythrocytes without reconstituted HA fusion protein (Control); RBC-HA: 5-Aza-2-dC-loaded erythro-magneto-HA virosomes.</p

FACS analysis of HeLa cells after (A) 24 h, (B) 48 h and (C) 96 h treatments.

<p><b>Ctrl</b> (control) untreated cells; <b>Aza:</b> cells treated with 1800 ng (2,5 µM) of 5-Aza-2-dC; <b>Erythro Aza:</b> cells treated with erythro-magneto-HA virosomes containing 180 ng of 5-Aza-2-dC; <b>Erythro:</b> unloaded 5-Aza-2-dC erythro-magneto-HA virosomes; <b>Supernatant:</b> cells treated with buffer where the erythro-magneto-HA virosomes were resuspended (control 2).</p

Confocal Laser Scanning Microscopy (CLSM).

<p>In the left is schematized the timing and mechanism of action of the erythro-magneto-HA virosomes, encapsulating 100 nm fluorescence-labelled magnetic nanoparticles (green) and 5-Aza-2-dC. In the right are shown CLSM images of erythro-magneto-HA virosomes (green) after 30 minutes (<b>A</b>), 1 hour (<b>B</b>), 6 hours (<b>C</b>), 24 and 96 hours (<b>D</b>) of incubation with HeLa cells highlighted by DAPI staining (blue). (<b>CTRL</b>) Untreated HeLa cells (control).</p

Engineered Erythrocytes.

<p>Confocal Laser Scanning Microscopy (CLSM) images of erytro-magneto-HA virosomes encapsulating 100 nm fluorescence-labelled superparamagnetic nanoparticles (green) and 5-Aza-2-dC.</p