W. Mark Saltzman

Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

Enhanced and prolonged cross‐presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles

CD8+ T‐cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8+ T cells against these cell‐associated antigens, exogenous antigens must be cross‐presented by professional antigen‐presenting cells (APCs). While cross‐presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein‐based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(d,l‐lactide‐co‐glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow‐derived dendritic cells (BMDCs), the MHC class I presentation of PLGA‐encapsulated ovalbumin (OVA) stimulated T cell interleukin‐2 secretion at 1000‐fold lower concentration than soluble antigen and 10‐fold lower than antigen‐coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead‐associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle‐mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

MicroRNA silencing for cancer therapy targeted to the tumour microenvironment

MicroRNAs are short non-coding RNAs expressed in different tissue and cell types that suppress the expression of target genes. As such, microRNAs are critical cogs in numerous biological processes, and dysregulated microRNA expression is correlated with many human diseases. Certain microRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumours that depend on these microRNAs are said to display oncomiR addiction. Some of the most effective anticancer therapies target oncogenes such as EGFR and HER2; similarly, inhibition of oncomiRs using antisense oligomers (that is, antimiRs) is an evolving therapeutic strategy. However, the in vivo efficacy of current antimiR technologies is hindered by physiological and cellular barriers to delivery into targeted cells. Here we introduce a novel antimiR delivery platform that targets the acidic tumour microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a non-endocytic pathway. We find that the attachment of peptide nucleic acid antimiRs to a peptide with a low pH-induced transmembrane structure (pHLIP) produces a novel construct that could target the tumour microenvironment, transport antimiRs across plasma membranes under acidic conditions such as those found in solid tumours (pH approximately 6), and effectively inhibit the miR-155 oncomiR in a mouse model of lymphoma. This study introduces a new model for using antimiRs as anti-cancer drugs, which can have broad impacts on the field of targeted drug delivery.

Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protected against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternate approach, using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA in the vaginal mucosa.

Building drug delivery into tissue engineering design

The creation of efficient methods for manufacturing biotechnology drugs — many of which influence fundamental but complex cell behaviours, such as proliferation, migration and differentiation — is creating new opportunities for tissue repair. Many agents are potent and multifunctional; that is, they produce different effects within different tissues. Therefore, control of tissue concentration and spatial localization of delivery is essential for safety and effectiveness. Synthetic systems that can control agent delivery are particularly promising as materials for enhancing tissue regeneration. This review discusses the state of the art in controlled-release and microfluidic drug delivery technologies, and outlines their potential applications for tissue engineering.

The uptake and intracellular fate of PLGA nanoparticles in epithelial cells

Biodegradable polymer nanoparticles (NPs) are a promising approach for intracellular delivery of drugs, proteins, and nucleic acids, but little is known about their intracellular fate, particularly in epithelial cells, which represent a major target. Rhodamine-loaded PLGA (polylactic-co-glycolic acid) NPs were used to explore particle uptake and intracellular fate in three different epithelial cell lines modeling the respiratory airway (HBE), gut (Caco-2), and renal proximal tubule (OK). To track intracellular fate, immunofluorescence techniques and confocal microscopy were used to demonstrate colocalization of NPs with specific organelles: early endosomes, late endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus. Confocal analysis demonstrated that NPs are capable of entering cells of all three types of epithelium. NPs appear to colocalize with the early endosomes at short times after exposure (approximately 2 h), but are also found in other compartments within the cytoplasm, notably Golgi and, possibly, ER, as time progressed over the period of 4-24 h. The rate and extent of uptake differed among these cell lines: at a fixed particle/cell ratio, cellular uptake was most abundant in OK cells and least abundant in Caco-2 cells. We present a model for the intracellular fate of particles that is consistent with our experimental data.

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.

Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery

Many synthetic polycationic vectors for non-viral gene delivery show high efficiency in vitro, but their usually excessive charge density makes them toxic for in vivo applications. Here we describe the synthesis of a series of high molecular weight terpolymers with low charge density, and show that they exhibit efficient gene delivery, some surpassing the efficiency of the commercial transfection reagents Polyethylenimine and Lipofectamine 2000. The terpolymers were synthesized via enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol, and their hydrophobicity adjusted by varying the lactone content and by selecting a lactone comonomer of specific ring size. Targeted delivery of the pro-apoptotic TRAIL gene to tumour xenografts by one of the terpolymers results in significant inhibition of tumour growth, with minimal toxicity both in vitro and in vivo. Our findings suggest that the gene delivery ability of the terpolymers stems from their high molecular weight and increased hydrophobicity, which compensates for their low charge density.

PEGylated PLGA nanoparticles for the improved delivery of doxorubicin

UNLABELLED We hypothesize that the efficacy of doxorubicin (DOX) can be maximized and dose-limiting cardiotoxicity minimized by controlled release from PEGylated nanoparticles. To test this hypothesis, a unique surface modification technique was used to create PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating DOX. An avidin-biotin coupling system was used to control poly(ethylene glycol) conjugation to the surface of PLGA nanoparticles, of diameter approximately 130 nm, loaded with DOX to 5% (wt/wt). Encapsulation in nanoparticles did not compromise the efficacy of DOX; drug-loaded nanoparticles were found to be at least as potent as free DOX against A20 murine B-cell lymphoma cells in culture and of comparable efficacy against subcutaneously implanted tumors. Cardiotoxicity in mice as measured by echocardiography, serum creatine phosphokinase (CPK), and histopathology was reduced for DOX-loaded nanoparticles as compared with free DOX. Administration of 18 mg/kg of free DOX induced a sevenfold increase in CPK levels and significant decreases in left ventricular fractional shortening over control animals, whereas nanoparticle-encapsulated DOX produced none of these pathological changes. FROM THE CLINICAL EDITOR The efficacy of doxorubicin (DOX) may be maximized and dose-limiting cardiotoxicity minimized by controlled release from PEGylated nanoparticles. Administration of 18 mg/kg of free DOX induced a sevenfold increase in CPK levels and significant decreases in left ventricular fractional shortening in mice, whereas nanoparticle-encapsulated DOX produced none of these pathological changes.

Distribution of nerve growth factor following direct delivery to brain interstitium

Several studies suggest the potential of nerve growth factor (NGF) in the treatment of patients with Alzheimers disease. To characterize NGF transport within the brain interstitium, we implanted controlled release polymers containing NGF and [125I]NGF into the brains of adult male rats and measured spatial distributions of NGF for up to one week. NGF concentration in the brain was quantified using ELISA, radiation counting, and autoradiography. At 2 days post-implantation, quantities of NGF in excess of 50 pg per section were detected within thick (1 mm) coronal slices of the hemisphere ipsilateral to the site of implantation up to 3 mm rostral and caudal to the edge of the polymer. Lower levels of radioactivity (> 5 pg but < 50 pg NGF per section) could be detected throughout the rest of the brain. Levels were highest in the tissue sections containing the polymer, reaching 9.5 ng per section. Autoradiography of thin (20 microns) coronal sections indicated that local NGF concentrations immediately adjacent to the polymer approached 40 micrograms/ml. Analysis of sequential sections on the autoradiograph confirmed that NGF was transported only 2-3 mm from the polymer in any direction. At one week post-implantation, the pattern of NGF distribution was similar to that seen at 2 days, and concentrations remained high near the site of the implant. Comparison of local NGF concentration profiles to simple models of diffusion with first-order elimination suggests that the NGF moved through the tissue by diffusion through the interstitial space with a half-life on the order of 0.5 h. The limited range of NGF transport in brain tissue indicates that: (i) protein drug agents such as NGF will probably need to be delivered almost directly to the site of action for efficacy; and (ii) toxicities associated with delivery of NGF and other protein agents to non-target cells, as often occurs with systemic delivery of drugs, may be reduced by local, interstitial delivery since therapy can be restricted to a small volume of the brain.