New approach to measuring oxygen diffusion and consumption in encapsulated living cells, based on electron spin resonance microscopy.

Cell microencapsulation within biocompatible polymers is an established technology for immobilizing living cells that secrete therapeutic products.  These can be transplanted into a desired site in the body for the controlled and continuous delivery of the therapeutic molecules.  One of the most important properties of the material that makes up the microcapsule is its oxygen penetrability, which is critical for the cells' survival.  Oxygen reaches the cells inside the microcapsules via a diffusion process.  The diffusion coefficient for the microcapsules' gel material is commonly measured using bulk techniques, where the gel in a chamber is first flushed with nitrogen and the subsequent rate of oxygen diffusion back into it is measured by an oxygen electrode placed in the chamber.  This technique does not address possible heterogeneities between microcapsules, and also cannot reveal O2 heterogeneity inside the microcapsule resulting from the living cells' activity.  Here we develop and demonstrate a proof of principle for a new approach to measuring and imaging the partial pressure of oxygen (pO2) inside a single microcapsule by means of high-resolution and high-sensitivity electron spin resonance (ESR).  The proposed methodology makes use of biocompatible paramagnetic microparticulates intercalated inside the microcapsule during its preparation.  The new ESR approach was used to measure the O2 diffusion properties of two types of gel materials (alginate and extracellular matrix - ECM), as well as to map a 3D image of the oxygen inside single microcapsules with living cells. STATEMENT OF SIGNIFICANCE: The technology of cell microencapsulation offers major advantages in the sustained delivery of therapeutic agents used for the treatment of various diseases ranging from diabetes to cancer. Despite the great advances made in this field, it still faces substantial challenges, preventing it from reaching the clinical practice. One of the primary challenges in developing cell microencapsulation systems is providing the cells with adequate supply of oxygen in the long term. Nevertheless, there is still no methodology good enough for measuring O2 distribution inside the microcapsule with sufficient accuracy and spatial resolution without affecting the microcapsule and/or the cells' activity in it. In the present work, we introduce a novel magnetic resonance technique to address O2 availability within cell-entrapping microcapsules. For the first time O2 distribution can be accurately measured and imaged within a single microcapsule. This new technique may be an efficient tool in the development of more optimal microencapsulation systems in the future, thus bringing this promising field closer to clinical application.

Innovative encapsulation platform based on pancreatic extracellular matrix achieve substantial insulin delivery.

Cell-based therapies for the treatment of diabetes, generally aim to provide long-term glucose regulated-insulin delivery using insulin producing cells. The delivery platform is crucial for the therapeutic outcome as well as for immunoisolation of the entrapped cells. We have developed a novel artificial pancreas encapsulation platform for the treatment of diabetes that is based on solubilized whole porcine pancreatic extracellular matrix (ECM). These unique capsules were used to entrap human liver cells and mesenchymal stem cells that were induced to differentiate into glucose-regulated insulin-producing cells. We demonstrate that the ECM-microcapsule platform provides a natural fibrous 3D niche, supporting cell viability and differentiation, while significantly improving insulin delivery. In vivo, ECM-encapsulated cells were shown to be non-immunogenic, and most importantly, to significantly improve the glycemic control in diabetic mouse preclinical model, thus establishing a proof-of-concept for this new cell-based insulin delivery platform.

Efficient transfection of tumors facilitated by long-term therapeutic ultrasound in combination with contrast agent: from in vitro to in vivo setting.

Therapeutic ultrasound (TUS) is a promising non-viral clinical approach for the delivery of genes. This study demonstrates the efficient delivery and localization of DNA in subcutaneous tumors facilitated by TUS application and examines the contribution of ultrasound contrast-agent (USCA) addition on transfection. The study addresses the importance of in vivo optimization when using long-term TUS and USCA based on data achieved in vitro. In vitro results showed that transfection of TrampC2 prostate cancer (Pca) cells using genes encoding for luciferase and green fluorescent protein was enhanced when DNA and Optison were added together and TUS was applied for 20 or 30 min. In vivo results showed that the highest transfection was achieved when Optison and DNA were co-injected intratumorally, and TUS was applied for 20 min. Using Optison significantly increased protein distribution in the tumor. However, in vivo expression level was decreased by two and four fold at 7 and 14 days, respectively, post-TUS. The study establishes the potential of intratumoral delivery of DNA-Optison, followed by TUS as an effective, non-toxic, gene delivery method that could provide a safe, clinical alternative to current viral gene delivery approaches where short-term gene expression is needed.

Therapeutic ultrasound-mediated DNA to cell and nucleus: bioeffects revealed by confocal and atomic force microscopy.

Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm(2), 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (>80%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.

Simultaneous inhibition of glioma angiogenesis, cell proliferation, and invasion by a naturally occurring fragment of human metalloproteinase-2.

Angiogenesis, tumor cell proliferation, and migration are the hallmarks of solid tumors, such as gliomas. This study demonstrates that a fragment derived from the autocatalytic digestion of matrix metalloproteinase (MMP)-2, called PEX, acts simultaneously as an inhibitor of glioma angiogenesis, cell proliferation, and migration. PEX is detected in the cultured medium of various human glioma, endothelial, breast, and prostate carcinoma cell lines. PEX is purified from the medium of glioma cell lines by chromatography, where PEX is constitutively expressed as a free and a TIMP-2-bound form. In human glioma tissue, PEX expression correlates with histological subtype and grade and with alpha v beta 3 integrin expression to which it is bound. Systemic administration of PEX to s.c. and intracranial human glioma xenografts results in a 99% suppression of tumor growth with no signs of toxicity. Thus, PEX is a very promising candidate for the treatment of human malignant gliomas.

Continuous release of endostatin from microencapsulated engineered cells for tumor therapy.

Research studies suggest that tumor-related angiogenesis contributes to the phenotype of malignant gliomas. We assessed the effect of local delivery of the angiogenesis inhibitor endostatin on human glioma cell line (U-87MG) xenografts. Baby hamster kidney (BHK) cells were stably transfected with a human endostatin (hES) expression vector and were encapsulated in alginate-poly L-lysine (PLL) microcapsules for long-term delivery of hES. The release of biologically active endostatin was confirmed using assays of bovine capillary endothelial (BCE) proliferation and of tube formation. Human endostatin released from the microcapsules brought about a 67. 2% inhibition of BCE proliferation. Furthermore, secreted hES was able to inhibit tube formation in KDR/PAE cells (porcine aortic endothelial cells stably transfected with KDR, a tyrosine kinase) treated with conditioned U-87MG medium. A single local injection of encapsulated endostatin-secreting cells in a nude mouse model resulted in a 72.3% reduction in subcutaneous U87 xenografts' weight 21 days post treatment. This inhibition was achieved by only 150.8 ng/ml human endostatin secreted from 2 x 10(5) encapsulated cells. Encapsulated endostatin-secreting cells are effective for the treatment of human glioblastoma xenografts. Continuous local delivery of endostatin may offer an effective therapeutic approach to the treatment of a variety of tumor types.

Bioreactors for decreasing the growth of brain tumors.

Malignant gliomas are the most common primary brain tumors. They are highly aggressive tumors characterized by a recurrence rate of virtually 100%. Despite significant advances in neuroimaging and neurosurgical techniques, the median survival time of patients with glioblastoma multiforme remains 12 to 18 months. Malignant gliomas are characterized by rapidly dividing cells, which invade into the normal brain, and a high degree of vascularity. Recent experimental evidence indicates that tumor-related angiogenesis contributes significantly to the malignant phenotype.

Enhancing the immunogenicity of liposomal hepatitis B surface antigen (HBsAg) by controlling its delivery from polymeric microspheres.

Microencapsulated liposome systems (MELs) were investigated as a potential immunization carrier for a recombinant 22-nm hepatitis B surface antigen (HBsAg) particle. MELs were prepared by first entrapping the HBsAg particles within liposomes composed of phosphatidylcholine:cholesterol (1:1 molar ratio), which were then encapsulated within alginate-poly(L-lysine) (PLL) hydrogel microspheres. The entrapped HBsAg particles retained immunoreactivity, as judged by an enzyme-linked immunosorbent assay (ELISA). Direct imaging of HBsAg particles and HBsAg incorporated into liposomes by cryo-transmission electron microscopy (cryo-TEM) indicated that HBsAg is embedded in the liposomal membrane. The antigenic particles were released from MELs mainly within the context of liposomes. The release rates in vitro and in vivo depended on the molecular weight of PLL used for MEL coating; MELs-214, coated with 214 kDa PLL, released the liposomal HBsAg at much higher rates than MELs-25, which was coated with 25 kDa PLL. Concomitantly, the specific anti-HBsAg titers in mice receiving HBsAg in MELs-214 were higher than those induced by MELs-25. MELs-214 were more efficient than conventional liposomes or alum in eliciting higher and prolonged antibody levels in mice. The ability of MELs to provide an HBsAg depot as well as a sustained release of liposomal HBsAg suggests that these carriers may be an ideal immunoadjuvant.

Controlled release of therapeutic agents: slow delivery and cell encapsulation.

Some of the most promising systems for the controlled release of bioactive agents, i.e., peptides or hormones, involve the encapsulation or entrapment of hormones or peptides in biocompatible polymeric devices that enable their continuous release over prolonged periods. In urology, two major pathologic conditions, androgen deficiency and prostate cancer, currently benefit from treatments based on controlled delivery. Leuprolide acetate depot (Lupron-depot) was one of the first controlled-delivery systems used for the treatment of prostate cancer. Clinical studies indicate that patients with prostate cancer who undergo therapy with leuprolide acetate depot can benefit from this treatment. Currently available androgen-replacement therapies include the oral administration of testosterone tablets or capsules, depot injections, sublingual treatment, and skin patches. However, side effects such as metabolic inactivation of testosterone on oral administration; fluctuations in levels of the hormone; and burning, rash, and skin necrosis during the use of skin patches may occur. These side effects may be avoided through the application of encapsulated Leydig cells, which produce testosterone. Studies in our laboratory have shown that Leydig cells encapsulated in alginate/poly-L-lysine/alginate microspheres are capable of secreting testosterone in culture and in vivo. Microencapsulated Leydig cells delivered intraperitoneally into castrated rats maintained a testosterone level of 0.51 ng/ml for more than 3 months without any human chorionic gonadotropin stimulation. Similar studies are also being conducted in our laboratory on encapsulation of ovarian cells for the secretion of progesterone and estrogen in culture and in vivo using microencapsulation techniques.

Ultrasonically enhanced transdermal drug delivery. Experimental approaches to elucidate the mechanism.

The effect of therapeutic range ultrasound on skin permeability was studied in vitro. Permeating molecule ionization state, pH, ultrasound duration, reversibility of the enhancement phenomenon, and skin structural alterations were evaluated. It was found that ultrasound affects the permeability of both ionized and unionized molecules. No irreversible structural alterations due to the ultrasound exposure were detected in the stratum corneum. Ultrasound enhancing mechanism was discussed.