María J. Blanco-Prieto

Sustained release of VEGF through PLGA microparticles improves vasculogenesis and tissue remodeling in an acute myocardial ischemia-reperfusion model.

The use of pro-angiogenic growth factors in ischemia models has been associated with limited success in the clinical setting, in part owing to the short lived effect of the injected cytokine. The use of a microparticle system could allow localized and sustained cytokine release and consequently a prolonged biological effect with induction of tissue revascularization. To assess the potential of VEGF(165) administered as continuous release in ischemic disease, we compared the effect of delivery of poly(lactic-co-glycolic acid) (PLGA) microparticles (MP) loaded with VEGF(165) with free-VEGF or control empty microparticles in a rat model of ischemia-reperfusion. VEGF(165) loaded microparticles could be detected in the myocardium of the infarcted animals for more than a month after transplant and provided sustained delivery of active protein in vitro and in vivo. One month after treatment, an increase in angiogenesis (small caliber caveolin-1 positive vessels) and arteriogenesis (α-SMA-positive vessels) was observed in animals treated with VEGF microparticles (p<0.05), but not in the empty microparticles or free-VEGF groups. Correlating with this data, a positive remodeling of the heart was also detected in the VEGF-microparticle group with a significantly greater LV wall thickness (p<0.01). In conclusion, PLGA microparticle is a feasible and promising cytokine delivery system for treatment of myocardial ischemia. This strategy could be scaled up and explored in pre-clinical and clinical studies.

Polymeric Particulates to Improve Oral Bioavailability of Peptide Drugs

Oral administration remains the most convenient way of delivering drugs. Recent advances in biotechnology have produced highly potent new molecules such as peptides, proteins and nucleic acids. Due to their sensitivity to chemical and enzymatic hydrolysis as well as a poor cellular uptake, their oral bioavailability remains very low. Despite sophisticated new delivery systems, the development of a satisfactory oral formulation remains a challenge. Among the possible strategies to improve the absorption of drugs, micro- and nanoparticles represent an exciting approach to enhance the uptake and transport of orally administered molecules. Increasing attention has been paid to their potential use as carriers for peptide drugs for oral administration. This article reviews the most common manufacturing methods for polymeric particles and the physiology of particle absorption from the gastrointestinal (GI) tract. In a second part, the use of polymeric particulate systems to improve the oral absorption of insulin is discussed.

Effective GDNF brain delivery using microspheres-A promising strategy for Parkinson’s disease

Glial cell line-derived neurotrophic factor (GDNF) has shown promise in the treatment of neurodegenerative disorders of basal ganglia origin such us Parkinsons disease (PD). In this study, we investigated the neurorestorative effect of controlled GDNF delivery using biodegradable microspheres in an animal model with partial dopaminergic lesion. Microspheres were loaded with N-glycosylated recombinant GDNF and prepared using the Total Recirculation One-Machine System (TROMS). GDNF-loaded microparticles were unilaterally injected into the rat striatum by stereotaxic surgery two weeks after a unilateral partial 6-OHDA nigrostriatal lesion. Animals were tested for amphetamine-induced rotational asymmetry at different times and were sacrificed two months after microsphere implantation for immunohistochemical analysis. The putative presence of serum IgG antibodies against rat glycosylated GDNF was analyzed for addressing safety issues. The results demonstrated that GDNF-loaded microspheres, improved the rotational behavior induced by amphetamine of the GDNF-treated animals together with an increase in the density of TH positive fibers at the striatal level. The developed GDNF-loaded microparticles proved to be suitable to release biologically active GDNF over up to 5 weeks in vivo. Furthermore, none of the animals developed antibodies against GDNF demonstrating the safety of glycosylated GDNF use.

Cytotoxicity of nanoscaled metal–organic frameworks

A series of fourteen porous Metal-Organic Frameworks (MOFs) with different compositions (Fe, Zn, and Zr; carboxylates or imidazolates) and structures have been successfully synthesised at the nanoscale and fully characterised by XRPD, FTIR, TGA, N2 porosimetry, TEM, DLS and ζ-potential. Their toxicological assessment was performed using two different cell lines: human epithelial cells from foetal cervical carcinoma (HeLa) and murine macrophage cell line (J774). It appears that MOF nanoparticles (NPs) exhibit low cytotoxicity, comparable to those of other commercialised nanoparticulate systems, the less toxic being the Fe carboxylate and the more toxic being the zinc imidazolate NPs. The cytotoxicity values, higher in J774 cells than in HeLa cells, are mainly function of their composition and cell internalisation capacity. Finally, cell uptake of one of the most relevant Fe-MOF-NPs for drug vectorisation has been investigated by confocal microscopy studies, and indicates a faster kinetics of cell penetration within J774 compared to HeLa cells.

PLGA microparticles: possible vehicles for topical drug delivery

Distribution of PLGA-microparticles in porcine skin, after its topical application, was studied in vitro using microparticles containing rhodamine as a fluorescent probe. PLGA-microparticles loaded with rhodamine were prepared using a solvent evaporation technique. Skin distribution of fluorescent microparticles was performed, by horizontal and vertical slicing of frozen skin. Fluorescence photomicrographs revealed that PLGA-microparticles could penetrate through the stratum corneum and reach the epidermis. However, permeation experiments showed that these microparticles were not able to reach the receptor compartment of the diffusion cells, even in a period of 24 h. The carriers described in this work could be used as vehicles for topical drug delivery, in order to obtain a sustained drug release into the skin, improving therapy by reduction of time intervals between doses.

Topical application of acyclovir-loaded microparticles : Quantification of the drug in porcine skin layers

The goal of this work was to increase the amount of acyclovir (ACV) in the basal epidermis, site of Herpes virus simplex infections, using microparticles as carriers. Poly(D,L-lactic-co-glycolic acid) microparticles loaded with ACV were prepared using a solvent evaporation technique. ACV distribution into porcine skin after topical application of microparticles for 6, 24 and 88 h, was determined by horizontal slicing of the skin. An ACV suspension served for comparison. The results showed that, at 6 and 24 h, the quantity of the drug in the basal epidermis with the microparticles, is similar to that obtained with the ACV suspension. However, after 88 h, the ACV reservoir in the basal epidermis was higher with the microparticles compared with the control suspension. This could be explained by the controlled drug release produced by the vector in the basal epidermis. Besides, at 88 h the amount of ACV detected in the receptor chamber of the diffusion cells was much lower with the microparticles than with the suspension. This type of carrier can improve acyclovir topical therapy since it increases drug retention in the basal epidermis and consequently increases the time intervals between doses.

Lipid raft-targeted therapy in multiple myeloma.

Despite recent advances in treatment, multiple myeloma (MM) remains an incurable malignancy. By using in vitro, ex vivo and in vivo approaches, we have identified here that lipid rafts constitute a new target in MM. We have found that the phospholipid ether edelfosine targets and accumulates in MM cell membrane rafts, inducing apoptosis through co-clustering of rafts and death receptors. Raft disruption by cholesterol depletion inhibited drug uptake by tumor cells as well as cell killing. Cholesterol replenishment restored MM cell ability to take up edelfosine and to undergo drug-induced apoptosis. Ceramide addition displaced cholesterol from rafts, and inhibited edelfosine-induced apoptosis. In an MM animal model, edelfosine oral administration showed a potent in vivo antimyeloma activity, and the drug accumulated preferentially and dramatically in the tumor. A decrease in tumor cell cholesterol, a major raft component, inhibited the in vivo antimyeloma action of edelfosine and reduced drug uptake by the tumor. The results reported here provide the proof-of-principle and rationale for further clinical evaluation of edelfosine and for this raft-targeted therapy to improve patient outcome in MM. Our data reveal cholesterol-containing lipid rafts as a novel and efficient therapeutic target in MM, opening a new avenue in cancer treatment.

In vitro and In vivo Selective Antitumor Activity of Edelfosine against Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia Involving Lipid Rafts

Purpose: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) remain B-cell malignancies with limited therapeutic options. The present study investigates the in vitro and in vivo effect of the phospholipid ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) in MCL and CLL. Experimental Design: Several cell lines, patient-derived tumor cells, and xenografts in severe combined immunodeficient mice were used to examine the anti-MCL and anti-CLL activity of edelfosine. Furthermore, we analyzed the mechanism of action and drug biodistribution of edelfosine in MCL and CLL tumor-bearing severe combined immunodeficient mice. Results: Here, we have found that the phospholipid ether edelfosine was the most potent alkyl-lysophospholipid analogue in killing MCL and CLL cells, including patient-derived primary cells, while sparing normal resting lymphocytes. Alkyl-lysophospholipid analogues ranked edelfosine > perifosine ≫ erucylphosphocholine ≥ miltefosine in their capacity to elicit apoptosis in MCL and CLL cells. Edelfosine induced coclustering of Fas/CD95 death receptor and rafts in MCL and CLL cells. Edelfosine was taken up by malignant cells, whereas normal resting lymphocytes hardly incorporated the drug. Raft disruption by cholesterol depletion inhibited drug uptake, Fas/CD95 clustering, and edelfosine-induced apoptosis. Edelfosine oral administration showed a potent in vivo anticancer activity in MCL and CLL xenograft mouse models, and the drug accumulated dramatically and preferentially in the tumor. Conclusions: Our data indicate that edelfosine accumulates and kills MCL and CLL cells in a rather selective way, and set coclustering of Fas/CD95 and lipid rafts as a new framework in MCL and CLL therapy. Our data support a selective antitumor action of edelfosine. Clin Cancer Res; 16(7); 2046–54. ©2010 AACR.

Controlled delivery of fibroblast growth factor-1 and neuregulin-1 from biodegradable microparticles promotes cardiac repair in a rat myocardial infarction model through activation of endogenous regeneration.

Acidic fibroblast growth factor (FGF1) and neuregulin-1 (NRG1) are growth factors involved in cardiac development and regeneration. Microparticles (MPs) mediate cytokine sustained release, and can be utilized to overcome issues related to the limited therapeutic protein stability during systemic administration. We sought to examine whether the administration of microparticles (MPs) containing FGF1 and NRG1 could promote cardiac regeneration in a myocardial infarction (MI) rat model. We investigated the possible underlying mechanisms contributing to the beneficial effects of this therapy, especially those linked to endogenous regeneration. FGF1- and NRG1-loaded MPs were prepared using a multiple emulsion solvent evaporation technique. Seventy-three female Sprague-Dawley rats underwent permanent left anterior descending coronary artery occlusion, and MPs were intramyocardially injected in the peri-infarcted zone four days later. Cardiac function, heart tissue remodeling, revascularization, apoptosis, cardiomyocyte proliferation, and stem cell homing were evaluated one week and three months after treatment. MPs were shown to efficiently encapsulate FGF1 and NRG1, releasing the bioactive proteins in a sustained manner. Three months after treatment, a statistically significant improvement in cardiac function was detected in rats treated with growth factor-loaded MPs (FGF1, NRG1, or FGF1/NRG1). The therapy led to inhibition of cardiac remodeling with smaller infarct size, a lower fibrosis degree and induction of tissue revascularization. Cardiomyocyte proliferation and progenitor cell recruitment were detected. Our data support the therapeutic benefit of NRG1 and FGF1 when combined with protein delivery systems for cardiac regeneration. This approach could be scaled up for use in pre-clinical and clinical studies.

Heart regeneration after myocardial infarction using synthetic biomaterials.

Myocardial infarction causes almost 7.3 million deaths each year worldwide. However, current treatments are more palliative than curative. Presently, cell and protein therapies are considered the most promising alternative treatments. Clinical trials performed until now have demonstrated that these therapies are limited by protein short half-life and by low transplanted cell survival rate, prompting the development of novel cell and protein delivery systems able to overcome such limitations. In this review we discuss the advances made in the last 10years in the emerging field of cardiac repair using biomaterial-based delivery systems with focus on the progress made on preclinical in vivo studies. Then, we focus in cardiac tissue engineering approaches, and how the incorporation of both cells and proteins together into biomaterials has opened new horizons in the myocardial infarction treatment. Finally, the ongoing challenges and the perspectives for future work in cardiac tissue engineering will also be discussed.