The Entrapment of Paclitaxel in PLGA Nanoparticles Increases its Cytotoxicity against Multiresistant Cell Line

Paclitaxel (Ptx) is a taxane anticancer mitotic inhibitor, widely used in oncology for the last 20 years. Poor solubility of Ptx, as a consequence using of toxic solvents such as Cremofor EL, high affinity to P-glycoprotein are associated with serious side effects due to hypersensitivity reactions, low bioavailability and low therapeutic index. Development of new delivery solvent-free forms of Ptx is one of the key research problems in modern cancer chemotherapy. Ptx loaded into polylactic-co-glycolic acid (PLGA) nanoparticles (Ptx-PLGA-Nps) (size 200-300 nm) have been prepared using nanoprecipitation method. Impact of technological parameters on Ptx encapsulation efficacy and in vitro drug release was investigated. Drug encapsulation was determined using HPLC. Citotoxic activity and cell accumulation of nanosomal formulation of Ptx was studied on multiresistant cell line Jurkat WT (cells of human Т-limphoblastic leucosis). Obtained results suggest that formulation of PLGA Ptx nanoparticles have above 90-98% drug encapsulation efficacy, higher cell accumulation and cytotoxic activity

Novel Potency Assay for MSC Secretome-Based Treatment of Idiopathic Male Infertility Employed Leydig Cells and Revealed Vascular Endothelial Growth Factor as a Promising Potency Marker

Idiopathic male infertility is a highly prevalent diagnosis in developed countries with no specific treatment options. Although empirical medical treatment is widely used to restore male fertility, its efficacy remains limited and inconclusively proven. Therefore, the development of novel therapeutic approaches in this field is a high-priority task. Since the failure of testicular microenvironment components might be involved in the pathogenesis of idiopathic male infertility, application of mesenchymal stromal cells (MSCs) as well as the MSC secretome is worth considering. Previously, we showed that the intratesticular injection of MSCs or the MSC secretome led to the recovery of spermatogenesis at least through replenishing the testicular microenvironment and its maintenance by MSC-secreted paracrine factors. However, the clinical use of such products has been limited to single trials to date. This may be due to the lack of relevant potency tests reflecting mechanisms of action of the MSC secretome in male infertility models. Based on the presumptive MSC secretome mode of action on the testicular microenvironment, we suggest a novel approach to test the potential efficacy of the MSC secretome for idiopathic male infertility treatment. It represents a potency assay based on evaluation of testosterone production by isolated Leydig cells. We demonstrated that the MSC secretome stimulated testosterone secretion by Leydig cells in vitro. We then hypothesized that among the major factors of the MSC secretome, vascular endothelial growth factor (VEGF) could be responsible for the observed effects, which we confirmed by the revealed correlation between the extent of stimulated testosterone production and VEGF concentration in the MSC secretome. The pilot results obtained from the doxorubicin-induced male infertility murine model also indicate the important impact of VEGF in the MSC secretome’s regenerative effects. Utilizing VEGF as a surrogate factor, a novel approach to study the potency of MSC secretome-based products for idiopathic male infertility treatment is suggested. Further validation is required for its implementation into the biopharmaceutical manufacturing process

Therapeutic Angiogenesis by a “Dynamic Duo”: Simultaneous Expression of HGF and VEGF165 by Novel Bicistronic Plasmid Restores Blood Flow in Ischemic Skeletal Muscle

Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene delivery was used to stimulate blood vessels’ formation and growth. We have previously shown that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse ischemic limb, and efficacy of combined delivery was superior to each plasmid administered alone. In this work, we evaluated different approaches for co-expression of HGF and VEGF165 genes in a panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional promoter or two independent promoters for each gene of interest. Studies in HEK293T culture showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of expressed factors. Tests in skeletal muscle explants showed a dramatic difference and most plasmids failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two independent promoters (cytomegalovirus (CMV) for HGF and chicken b-actin (CAG) for VEGF165) provided expression of both grow factors in skeletal muscle at an equimolar ratio. Efficacy tests of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular administration of plasmid induced significant restoration of perfusion compared to an empty vector and saline. These findings were supported by increased CD31+ capillary density in animals that received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide basis for future development of treatment for an unmet medical need—peripheral artery disease and associated limb ischemia

Exploring the Interplay between Drug Release and Targeting of Lipid-Like Polymer Nanoparticles Loaded with Doxorubicin

Targeted delivery of doxorubicin still poses a challenge with regards to the quantities reaching the target site as well as the specificity of the uptake. In the present approach, two colloidal nanocarrier systems, NanoCore-6.4 and NanoCore-7.4, loaded with doxorubicin and characterized by different drug release behaviors were evaluated in vitro and in vivo. The nanoparticles utilize a specific surface design to modulate the lipid corona by attracting blood-borne apolipoproteins involved in the endogenous transport of chylomicrons across the blood–brain barrier. When applying this strategy, the fine balance between drug release and carrier accumulation is responsible for targeted delivery. Drug release experiments in an aqueous medium resulted in a difference in drug release of approximately 20%, while a 10% difference was found in human serum. This difference affected the partitioning of doxorubicin in human blood and was reflected by the outcome of the pharmacokinetic study in rats. For the fast-releasing formulation NanoCore-6.4, the AUC0→1h was significantly lower (2999.1 ng × h/mL) than the one of NanoCore-7.4 (3589.5 ng × h/mL). A compartmental analysis using the physiologically-based nanocarrier biopharmaceutics model indicated a significant difference in the release behavior and targeting capability. A fraction of approximately 7.310–7.615% of NanoCore-7.4 was available for drug targeting, while for NanoCore-6.4 only 5.740–6.057% of the injected doxorubicin was accumulated. Although the targeting capabilities indicate bioequivalent behavior, they provide evidence for the quality-by-design approach followed in formulation development

Urokinase-Type Plasminogen Activator Enhances the Neuroprotective Activity of Brain-Derived Neurotrophic Factor in a Model of Intracerebral Hemorrhage

Brain-derived neurotrophic factor (BDNF) is a classic neuroprotective and pro-regenerative factor in peripheral and central nervous tissue. Its ability to stimulate the restoration of damaged nerve and brain tissue after ischemic stroke and intraventricular hemorrhage has been demonstrated. However, the current concept of regeneration allows us to assert that one factor, even if essential, cannot be the sole contributor to this complex biological process. We have previously shown that urokinase-type plasminogen activator (uPA) complements BDNF activity and stimulates restoration of nervous tissue. Using a model of intracerebral hemorrhage in rats, we investigated the neurotrophic and neuroprotective effect of BDNF combined with uPA. The local simultaneous administration of BDNF and uPA provided effective neuroprotection of brain tissue after intracerebral hemorrhage, promoted survival of experimental animals and their neurological recovery, and decreased lesion volume. The study of cellular mechanisms of the observed neurotrophic effect of BDNF and uPA combination revealed both known mechanisms (neuronal survival and neurite growth) and new ones (microglial activation) that had not been shown for BDNF and uPA. Our findings support the concept of using combinations of biological factors with diverse but complementary mechanisms of action as a promising regenerative approach