Intra-amniotic Injection of Poly(lactic-co-glycolic Acid) Microparticles Loaded with Growth Factor: Effect on Tissue Coverage and Cellular Apoptosis in the Rat Model of Myelomeningocele.

BACKGROUND: Myelomeningocele (MMC) is a devastating congenital neurologic disorder that can lead to lifelong morbidity and has limited treatment options. This study investigates the use of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) loaded with fibroblast growth factor (FGF) as a platform for in utero treatment of MMC. STUDY DESIGN: Intra-amniotic injections of PLGA MPs were performed on gestational day 17 (E17) in all-trans retinoic acid-induced MMC rat dams. MPs loaded with fluorescent dye (DiO) were evaluated 3 hours after injection to determine incidence of binding to the MMC defect. Fetuses were then treated with PBS or PLGA particles loaded with DiO, bovine serum albumin, or FGF and evaluated at term (E21). Fetuses with MMC defects were evaluated for gross and histologic evidence of soft tissue coverage. The effect of PLGA-FGF treatment on spinal cord cell death was evaluated using an in situ cell death kit. RESULTS: PLGA-DiO MPs had a binding incidence of 86% and 94% 3 hours after injection at E17 for doses of 0.1 mg and 1.2 mg, respectively. Incidence of soft tissue coverage at term was 19% (4 of 21), 22% (2 of 9), and 83% (5 of 6) for PLGA-DiO, PLGA-BSA, and PLGA-FGF, respectively. At E21, the percentage of spinal cord cells positive for in situ cell death was significantly higher in MMC controls compared with wild-type controls or MMC pups treated with PLGA-FGF. CONCLUSION: PLGA MPs are an innovative minimally invasive platform for induction of soft tissue coverage in the rat model of MMC and may reduce cellular apoptosis.

Intrathecal delivery and its applications in leptomeningeal disease.

Intrathecal delivery (IT) of opiates into the cerebrospinal fluid (CSF) for anesthesia and pain relief has been used clinically for decades, but this relatively straightforward approach of bypassing the blood-brain barrier has been underutilized for other indications because of its lack of utility in delivering small lipid-soluble drugs. However, emerging evidence suggests that IT drug delivery be an efficacious strategy for the treatment of cancers in which there is leptomeningeal spread of disease. In this review, we discuss CSF flow dynamics and CSF clearance pathways in the context of intrathecal delivery. We discuss human and animal studies of several new classes of therapeutic agents-cellular, protein, nucleic acid, and nanoparticle-based small molecules-that may benefit from IT delivery. The complexity of the CSF compartment presents several key challenges in predicting biodistribution of IT-delivered drugs. New approaches and strategies are needed that can overcome the high rates of turnover in the CSF to reach specific tissues or cellular targets.

Nanoparticle-mediated convection-enhanced delivery of a DNA intercalator to gliomas circumvents temozolomide resistance.

In patients with glioblastoma, resistance to the chemotherapeutic temozolomide (TMZ) limits any survival benefits conferred by the drug. Here we show that the convection-enhanced delivery of nanoparticles containing disulfide bonds (which are cleaved in the reductive environment of the tumour) and encapsulating an oxaliplatin prodrug and a cationic DNA intercalator inhibit the growth of TMZ-resistant cells from patient-derived xenografts, and hinder the progression of TMZ-resistant human glioblastoma tumours in mice without causing any detectable toxicity. Genome-wide RNA profiling and metabolomic analyses of a glioma cell line treated with the cationic intercalator or with TMZ showed substantial differences in the signalling and metabolic pathways altered by each drug. Our findings suggest that the combination of anticancer drugs with distinct mechanisms of action with selective drug release and convection-enhanced delivery may represent a translational strategy for the treatment of TMZ-resistant gliomas.

Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells.

Human endothelial cells are initiators and targets of the rejection response. Pre-operative modification of endothelial cells by small interfering RNA transfection could shape the nature of the host response post-transplantation. Ablation of endothelial cell class II major histocompatibility complex molecules by small interfering RNA targeting of class II transactivator can reduce the capacity of human endothelial cells to recruit and activate alloreactive T cells. Here, we report the development of small interfering RNA-releasing poly(amine-co-ester) nanoparticles, distinguished by their high content of a hydrophobic lactone. We show that a single transfection of small interfering RNA targeting class II transactivator attenuates major histocompatibility complex class II expression on endothelial cells for at least 4 to 6 weeks after transplantation into immunodeficient mouse hosts. Furthermore, silencing of major histocompatibility complex class II reduces allogeneic T-cell responses in vitro and in vivo. These data suggest that poly(amine-co-ester) nanoparticles, potentially administered during ex vivo normothermic machine perfusion of human organs, could be used to modify endothelial cells with a sustained effect after transplantation.The use of gene silencing techniques in the treatment of post-transplantation host rejection is not long lasting and can have systemic effects. Here, the authors utilize a nanocarrier for siRNA for treatment of arteries ex vivo prior to implantation subsequently attenuating immune reaction in vivo.

In vivo correction of anaemia in ß-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery.

The blood disorder, ß-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human ß-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% ß-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.

Surface-modified nanoparticles enhance transurothelial penetration and delivery of survivin siRNA in treating bladder cancer.

Penetration of the bladder permeability barrier (BPB) is a major challenge when treating bladder diseases via intravesical delivery. To increase transurothelial migration and tissue and tumor cell uptake, poly(lactic-co-glycolic acid; PLGA) nanoparticles (NP) were modified by addition of a low molecular weight (2.5 or 20 kDa) positively charged mucoadhesive polysaccharide, chitosan, to the NP surface. In designing these NPs, we balanced the adhesive properties of chitosan with the release and bioactivity of the siRNA. Chitosan-functionalized NPs demonstrated increased binding to and uptake in intravesically instilled mouse bladders and human ureter at 10 times the level of unmodified NPs. Furthermore, we extended the bioactivity of survivin siRNA in vitro for up to 9 days and demonstrated a decrease in proliferation when using chitosan-modified NPs relative to unmodified NPs. In addition, treatment of xenograft tumors with chitosan-modified NPs that encapsulate survivin siRNA (NP-siSUR-CH2.5) resulted in a 65% reduction in tumor volume and a 75% decrease in survivin expression relative to tumors treated with blank chitosan NPs (NP-Bk-CH2.5). Our low molecular weight chitosan delivery system has the capacity to transport large amounts of siRNA across the urothelium and/or to the tumor site, thus increasing therapeutic response.

Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo.

In vivo delivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD.Cg-Prkdc(scid)IL2rγ(tm1Wjl) (abbreviated NOD-scid IL2rγ(null)) mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the ß-globin gene using nanoparticles carrying ß-globin-targeted PNAs/DNAs, demonstrating this method's versatility. In vivo testing in an enhanced green fluorescent protein-ß-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy.

Innate immune system activation is a critical step in the initiation of an effective adaptive immune response; therefore, activation of a class of innate pathogen receptors called pattern recognition receptors (PRR) is a central feature of many adjuvant systems. It has recently been shown that one member of an intracellular PRR, the NLRP3 inflammasome, is activated by a number of classical adjuvants including aluminum hydroxide and saponins [Eisenbarth SC, Colegio OR, O'Connor W, Sutterwala FS, Flavell RA. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 2008;453(June (7198)):1122-6; Li H, Willingham SB, Ting JP, Re F. Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3. J Immunol 2008;181(July (1)):17-21]. Inflammasome activation in vitro requires signaling of both the Toll-like receptor (TLR) and NLRP3 in antigen-presenting cells. Here we present a class of nanomaterials endowed with these two signals for rapid optimization of vaccine design. We constructed this system using a simple approach that incorporates lipopolysaccharides (LPS) onto the surface of nanoparticles constructed from a biocompatible polyester, poly(lactic-co-glycolic acid) (PLGA), loaded with antigen. We demonstrate that LPS-modified particles are preferentially internalized by dendritic cells compared to uncoated nanoparticles and the system, when administered to mice, elicits potent humoral and cellular immunity against a model antigen, ovalbumin. Wild-type macrophages pulsed with LPS-modified nanoparticles resulted in production of the proinflammatory cytokine IL-1beta consistent with inflammasome activation. In comparison, NLRP3-deficient and caspase-1-deficient macrophages showed negligible production of IL-1beta. Furthermore, when endocytosis and lysosomal destabilization were inhibited, inflammasome activity was diminished, supporting the notion that nanoparticles rupture lysosomal compartments and behave as 'danger signals' [Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008;9(August (8)):847-56]. The generality of this vaccination approach is tested by encapsulation of a recombinant West Nile envelope protein and demonstrated by protection against a murine model of West Nile encephalitis. The design of such an antigen delivery mechanism with the ability to stimulate two potent innate immune pathways represents a potent new approach to simultaneous antigen and adjuvant delivery.

Conjugation to increase treatment volume during local therapy: a case study with PEGylated camptothecin.

Controlled release of chemotherapy drugs from polymer implants placed directly at the tumor site is a proven method for treatment of cancers of the brain. Although this method provides high doses of drug at the tumor site, the drug does not penetrate far enough into the brain for optimum treatment in most cases. Rapid drug elimination leads to more than a 10-fold drop in concentration within 2 mm of the implant. Conjugation to water-soluble polymers, such as poly(ethylene glycol) (PEG) or dextran, has the potential to increase drug distribution in the brain. We have recently PEGylated the chemotherapy drug camptothecin and found a large increase in the extent of distribution of camptothecin in the rat brain, but most of the drug in tissue was in the less-active conjugated form. Stability of the conjugation bond, activity of the drug-polymer conjugate, solubility of the conjugate relative to the drug, and molecular weight of the polymer must all be considered in the design of a conjugate to maximize drug distribution. Therefore, to optimize the PEGylated system, we have developed a pharmacokinetic model to determine the relative importance of parameters involved in the distribution of drug-polymer conjugates after release from a polymer implant. Our modeling shows that PEGylation has the potential to increase treatment distances to more than a centimeter, which may be sufficient to prevent the recurrence of human brain tumors.

A PEDF N-terminal peptide protects the retina from ischemic injury when delivered in PLGA nanospheres.

The neuroprotective effects of small pigment epithelium-derived factor (PEDF) peptides injected intravitreally as free peptides or delivered in poly(lactide-co-glycolide) (PLGA) nanospheres, were tested in retinal ischemic injury. We induced transient ischemia in C57BL/6 mice by elevating the intraocular pressure to the equivalent of 120 mmHg for 60 min, then injected these eyes with one of the following: PBS, full-length native PEDF, N-terminal peptides-PEDF(136-155) and PEDF(82-121), blank PLGA nanospheres or PLGA loaded with PEDF(82-121) (PLGA-PEDF(82-121)). Morphometric analysis and TUNEL assays were used to determine the extent of retinal damage. Transient ischemia caused a rapid reduction in the number of viable cells in the retinal ganglion cell (RGC) layer over 48h as compared to non-ischemic retinas. About 76% surviving cells in the RGC layer were observed in the full-length PEDF protein treated group, whereas only 32% of cells survived in the PBS group. Thus, PEDF prevented approximately 44% of the cell death in the RGC layer resulting from transient ischemia. PEDF(82-121) peptide was as effective as full-length PEDF when injected as either a free peptide or delivered in PLGA nanospheres. PLGA-PEDF(82-121) showed longer-term protection of the RGC layer with no noticeable side effects at 7days. PEDF and PEDF(82-121) lessened damage to the IPL as measured by layer thickness. PEDF and PEDF(82-121) also delayed retinal responses to ischemic injury as measured by GFAP immunolabeling in Müller cells. PEDF(82-121) is an effective neuroprotective peptide in retinal ischemia. PLGA-PEDF(82-121) offers greater protection to the retina suggesting that this peptide and the method of delivering therapeutically active drugs have potential clinical advantages for longer-term treatments of retinal diseases.

Surface modification of biodegradable polyesters with fatty acid conjugates for improved drug targeting.

We describe a general method for incorporating target ligands into the surface of biocompatible polyester poly(lactic-co-glycolic acid) (PLGA) 50/50 materials using fatty acids. Avidin-fatty acid conjugates were prepared and efficiently incorporated into PLGA. Avidin was chosen as an adaptor protein to facilitate the attachment of a variety of biotinylated ligands. We show that fatty acid preferentially associates with the hydrophobic PLGA matrix, rather than the external aqueous environment, facilitating a prolonged presentation of avidin over several weeks. We successfully applied this approach in both microspheres encapsulating a model protein, bovine serum albumin, and PLGA scaffolds fabricated by a salt-leaching method. Because of its ease, generality and flexibility, this strategy promises widespread utility in modifying the surface of PLGA-based materials for applications in drug delivery and tissue engineering.

Biomimetic design in microparticulate vaccines.

Current efforts to improve the effectiveness of microparticle vaccines include incorporating biomimetic features into the particles. Many pathogens use surface molecules to target specific cell types in the gut for host invasion. This observation has inspired efforts to chemically conjugate cell-type targeting ligands to the surfaces of microparticles in order to increase the efficiency of uptake, and therefore the effectiveness, of orally administered microparticles. Bio-mimicry is not limited to the exterior surface of the microparticles. Anti-idiotypic antibodies, cytokines or other biological modifiers can be encapsulated for delivery to sites of interest as vaccines or other therapeutics. Direct mucosal delivery of microparticle vaccines or immunomodulatory agents may profoundly enhance mucosal and systemic immune responses compared to other delivery routes.

Materials for protein delivery in tissue engineering.

The ability of protein agents to modulate cellular behaviors, such as motility, proliferation, adhesion and function, is the subject of intense research; new therapies involving proteins will likely result. Unfortunately, many proteins have short half-lives and the potential for toxicity after systemic delivery, so traditional routes of administration are not appropriate. Alternate methods for sustained delivery of these agents to the desired cells and tissues in biologically active conformations and concentrations are necessary. Techniques similar to those long used in the controlled delivery of drugs have been used to administer certain growth factors to cells and tissues; although clinical success has been limited to date, studies in animal models suggest the potential for tremendous advances in the near future. This review outlines the basic technology of controlled protein delivery using polymeric materials, and discusses some of the techniques under investigation for the efficient administration of proteins in tissue engineering.

Cell aggregation and neurite growth in gels of extracellular matrix molecules.

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.