Nanodelivery of doxorubicin for age-related macular degeneration

Objective: Polymeric nanoparticles (NPs) containing doxorubicin (DOX) were prepared for the inhibition of hypoxia-induced factor 1α (HIF-1α).

Significance: HIF-1α is responsible for the upregulation of several angiogenic factors, including vascular endothelial growth factor (VEGF). DOX inhibits HIF-1α but is highly toxic. By encapsulating DOX in NPs, drug delivery will be sustained and toxicity will be reduced without limiting efficacy.

Methods: DOX NPs were prepared using both polylactic coglycolic acid (PLGA) and chitosan. PLGA NPs were prepared via nanoprecipitation (NPC) and single and double emulsion diffusion (SE; DE). Chitosan NPs were formulated using ionic gelation (IG), and complex coacervation (CC). Size, polydispersity index (PDI), and zeta potential (ZP) were determined via dynamic light scattering (DLS) (n?=?3). The encapsulation efficiency (EE), drug loading capacity (DLC) (n?=?3) and in vitro drug release profiles (IVR) at 37?°C (n?=?4) were analyzed via spectroscopy at 480?nm (λ_max). The cytotoxicity of each formulation as well as free DOX solution in ARPE-19 cells was determined via MTT assay after 24?h (n?=?3). HIF-1α and VEGF inhibition in ARPE-19 cells were measured via ELISA (n?=?3).

Results: The results were consistent with the hypothesis; the NP formulations decreased HIF-1α and VEGF-A expression in ARPE-19 cells with reduced cytotoxicity. SE, DE, and CC demonstrated low ZP as well as the most rapid drug release of the tested formulations. FTIR confirmed the presence of DOX on the SE NP surface, indicating instability.

Conclusions: SE, DE, and CC destabilized. NPC was the most efficient formulation for the nanodelivery of DOX for AMD.     

Camouflaged Hybrid Cancer Cell-Platelet Fusion Membrane Nanovesicles Deliver Therapeutic MicroRNAs to Presensitize Triple-Negative Breast Cancer to Doxorubicin

Camouflaged cell-membrane-based nanoparticles have gained increasing attention owing to their improved biocompatibility and immunomodulatory properties. Using nanoparticles prepared from the membranes of specific cell types or fusions derived from different cells membranes, their functional performance could be improved in several aspects. Here, cell membranes extracted from breast cancer cells and platelets are used to fabricate a hybrid-membrane vesicle (cancer cell-platelet-fusion-membrane vesicle, CPMV) loaded with therapeutic microRNAs (miRNAs) for the treatment of triple-negative breast cancer (TNBC). A clinically scalable microfluidic platform is presented for fusion of cell membranes. The reconstitution process during synthesis allows for efficient loading of miRNAs into CPMVs. Conditions for preparation of miRNA-loaded CPMVs are systematically optimized and their property of homing to source cells is demonstrated using in vitro experiments and therapeutic evaluation in vivo. In vitro, the CPMVs exhibit significant recognition of their source cells and avoided engulfment by macrophages. After systemic delivery in mice, CPMVs show a prolonged circulation time and site-specific accumulation at implanted TNBC-xenografts. The delivered antimiRNAs are sensitized TNBCs to doxorubicin, resulting in an improved therapeutic response and survival rate. This strategy has considerable potential for clinical translation to improve personalized therapy for breast cancer and other malignancies.     

Mixed Nanosized Polymeric Micelles as Promoter of Doxorubicin and miRNA‐34a Co‐Delivery Triggered by Dual Stimuli in Tumor Tissue

Dual stimuli‐sensitive mixed polymeric micelles (MM) are developed for co‐delivery of the endogenous tumor suppressor miRNA‐34a and the chemotherapeutic agent doxorubicin (Dox) into cancer cells. The novelty of the system resides in two stimuli‐sensitive prodrugs, a matrix metalloproteinase 2 (MMP2)‐sensitive Dox conjugate and a reducing agent (glutathione, GSH)‐sensitive miRNA‐34a conjugate, self‐assembled in a single particle decorated with a polyethylene glycol corona for longevity, and a cell‐penetrating peptide (TATp) for enhanced intracellular delivery. The MMP2‐sensitivity of the system results in threefold higher cytotoxicity in MMP2‐overexpressing HT1080 cells compared to low MMP2‐expressing MCF7 cells. Cellular internalization of Dox increases by more than 70% after inclusion of TATp to the formulation. MMP2‐sensitive MM also inhibits proliferation and migration of HT1080 cells. Moreover, GSH‐sensitive MM allows for an efficient downregulation of Bcl2, survivin, and notch1 (65%, 55%, and 46%, respectively) in HT1080 cells. Combination of both conjugates in dual sensitive MM reduces HT1080 cell viability to 40% and expression of Bcl2 and survivin. Finally, 50% cell death is observed in 3D models of tumor mass. The results confirm the potential of the MM to codeliver miRNA‐34a and doxorubicin triggered by dual stimuli inherent of tumor tissues.     

The Combination of the M2 Muscarinic Receptor Agonist and Chemotherapy Affects Drug Resistance in Neuroblastoma Cells

One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.     

Elevated Production of Mitochondrial Reactive Oxygen Species via Hyperthermia Enhanced Cytotoxic Effect of Doxorubicin in Human Breast Cancer Cell Lines MDA-MB-453 and MCF-7

Hyperthermia (HT) treatment is a noninvasive cancer therapy, often used with radiation therapy and chemotherapy. Compared with 37 °C, 42 °C is mild heat stress for cells and produces reactive oxygen species (ROS) from mitochondria. To involve subsequent intracellular accumulation of DOX, we have previously reported that the expression of ATP-binding cassette sub-family G member 2 (ABCG2), an exporter of doxorubicin (DOX), was suppressed by a larger amount of intracellular mitochondrial ROS. We then hypothesized that the additive effect of HT and chemotherapy would be induced by the downregulation of ABCG2 expression via intracellular ROS increase. We used human breast cancer cell lines, MCF-7 and MDA-MB-453, incubated at 37 °C or 42 °C for 1 h to clarify this hypothesis. Intracellular ROS production after HT was detected via electron spin resonance (ESR), and DOX cytotoxicity was calculated. Additionally, ABCG2 expression in whole cells was analyzed using Western blotting. We confirmed that the ESR signal peak with HT became higher than that without HT, indicating that the intracellular ROS level was increased by HT. ABCG2 expression was downregulated by HT, and cells were injured after DOX treatment. DOX cytotoxicity enhancement with HT was considered a result of ABCG2 expression downregulation via the increase of ROS production. HT increased intracellular ROS production and downregulated ABCG2 protein expression, leading to cell damage enhancement via DOX.     

Modification of Magnetite Cellulose Nanoparticles via Click Reaction for use in Controlled Drug Delivery

Superparamagnetic Fe₃O₄ nanoparticles (MNPs) were functionalized by modified cellulose. The modified cellulose was synthesized through bromoacetylation of cellulose (BACell) followed by the substitution of sodium azide to form BACell-N₃. The remaining methylene bromide groups on BACell-N₃ was further reacted with the MNPs to form Fe₃O₄/Cell-N₃. Then propargyl alcohol (PA) was immobilized on the azide-terminated Fe₃O₄ nanoparticles through copper (I)-catalyzed azide-alkyne cycloaddition (click reaction) to form Fe₃O₄/Cell/TAA nanoparticles. Doxorubicin (DOX) was loaded on prepared nanoparticles and release profiles of the DOX as a model drug from the Fe₃O₄/Cell/TAA nanoparticles and its loading capacity were determined by UV–Vis absorption at λ_max 483?nm.