No abstract

The direct local delivery of short interfering RNA (siRNA) to tissues may present solutions to several complex medical conditions. In particular, chronic wound healing is a serious and painful complication of diabetes mellitus (DM) affecting as many as one in four patients with a three year recurrence rate of more than 50% and leaving over 70,000 patients in the United States alone facing amputation. Here we describe the use of siRNA delivered locally into the diabetic ulcer directly and in a sustained fashion to knockdown a chronically upregulated extracellular matrix protease, matrix metalloproteinase-9 (MMP-9), to improve wound healing.

Selective isolation and purification of circulating tumor cells (CTCs) from whole blood is an important capability for both clinical medicine and biological research. Current techniques to perform this task place the isolated cells under excessive stresses that reduce cell viability, and potentially induce phenotype change, therefore losing valuable information about the isolated cells. We present a biodegradable nano-film coating on the surface of a microfluidic chip, which can be used to effectively capture as well as non-invasively release cancer cell lines such as PC-3, LNCaP, DU 145, H1650 and H1975. We have applied layer-by-layer (LbL) assembly to create a library of ultrathin coatings using a broad range of materials through complementary interactions. By developing an LbL nano-film coating with an affinity-based cell-capture surface that is capable of selectively isolating cancer cells from whole blood, and that can be rapidly degraded on command, we are able to gently isolate cancer cells and recover them without compromising cell viability or proliferative potential. Our approach has the capability to overcome practical hurdles and provide viable cancer cells for downstream analyses, such as live cell imaging, single cell genomics, and in vitro cell culture of recovered cells. Furthermore, CTCs from cancer patients were also captured, identified, and successfully released using the LbL-modified microchips.

Layer-by-layer (LbL) assembly is a powerful tool with increasing real world applications in energy, biomaterials, active surfaces, and membranes; however, the current state of the art requires individual sample construction using large quantities of material. Here we describe a technique using capillary flow within a microfluidic device to drive high-throughput assembly of LbL film libraries. This capillary flow layer-by-layer (CF-LbL) method significantly reduces material waste, improves quality control, and expands the potential applications of LbL into new research spaces. The method can be operated as a simple lab benchtop apparatus or combined with liquid-handling robotics to extend the library size. Here we describe and demonstrate the technique and establish its ability to recreate and expand on the known literature for film growth and morphology. We use the same platform to assay biological properties such as cell adhesion and proliferation and ultimately provide an example of the use of this approach to identify LbL films for surface-based DNA transfection of commonly used cell types.

No abstract

The success of RNA interference (RNAi) in medicine relies on the development of technology capable of successfully delivering it to tissues of interest. Significant research has focused on the difficult task of systemic delivery of RNAi; however its local delivery could be a more easily realized approach. Localized delivery is of particular interest for many medical applications, including the treatment of localized diseases, the modulation of cellular response to implants or tissue engineering constructs, and the management of wound healing and regenerative medicine. In this work we present an ultra-thin electrostatically assembled coating for localized and sustained delivery of short interfering RNA (siRNA). This film was applied to a commercially available woven nylon dressing commonly used for surgical applications, and was demonstrated to sustain significant knockdown of protein expression in multiple cell types for over one week in vitro. Significantly, this coating can be easily applied to a medically relevant device and requires no externally delivered transfection agents for effective delivery of siRNA. These results present promising opportunities for the localized administration of RNAi.

Wound healing is an incredibly complex biological process that often results in thickened collagen-enriched healed tissue called scar. Cutaneous scars lack many functional structures of the skin such as hair follicles, sweat glands, and papillae. The absence of these structures contributes to a number of the long-term morbidities of wound healing, including loss of function for tissues, increased risk of re-injury, and aesthetic complications. Scar formation is a pervasive factor in our daily lives; however, in the case of serious traumatic injury, scars can create long-lasting complications due to contraction and poor tissue remodeling. Within this report we target the expression of connective tissue growth factor (CTGF), a key mediator of TGFβ pro-fibrotic response in cutaneous wound healing, with controlled local delivery of RNA interference. Through this work we describe both a thorough in vitro analysis of nanolayer coated sutures for the controlled delivery of siRNA and its application to improve scar outcomes in a third-degree burn induced scar model in rats. We demonstrate that the knockdown of CTGF significantly altered the local expression of αSMA, TIMP1, and Col1a1, which are known to play roles in scar formation. The knockdown of CTGF within the healing burn wounds resulted in improved tissue remodeling, reduced scar contraction, and the regeneration of papillary structures within the healing tissue. This work adds support to a number of previous reports that indicate CTGF as a potential therapeutic target for fibrosis. Additionally, we believe that the controlled local delivery of siRNA from ultrathin polymer coatings described within this work is a promising approach in RNA interference that could be applied in developing improved cancer therapies, regenerative medicine, and fundamental scientific research.

Uncontrolled bleeding and infection are the major causes of death and morbidity from traumatic wounds during military conflicts, disasters, and accidents. Because immediate treatment is critical to survival, it is desirable to have a lightweight and rapidly applicable bandageone capable of delivering a hemostat that can quickly resolve bleeding while addressing infection over short and longer time frames. It is challenging to design thin film coatings capable of multidrug release, particularly when the drugs are quite different in nature (biologic versus small molecule, charged versus neutral) and the desired release profiles are different for each drug. Herein we have adopted a layer-by-layer film assembly technique to create a linear combination of two independently functional films capable of rapidly releasing thrombin within minutes while sustaining vancomycin elution for more than 24 h. By conjugating vancomycin to a hydrolytically degradable polyacid, poly(β-L-malic acid), we were able to create a robust thin film with loading and release kinetics that remain unaffected by the additional deposition of a thrombin-based film, demonstrating the possibility for future multitherapeutic films with independently tunable release kinetics.