Pigment epithelium-derived factor engineered to increase glycosaminoglycan affinity while maintaining bioactivity.

Pigment epithelium-derived factor (PEDF) is a secreted protein that is essential in tissue homeostasis and is involved in multiple functions in the eye, such as antiangiogenesis and neuroprotection. However, short retention in the retinal microenvironment can limit its therapeutic effects. In this study, we modified the amino acid sequence of PEDF to increase its affinity for heparin and hyaluronic acid (HA), which are negatively charged extracellular matrix (ECM) molecules. HA is the major component of the vitreous humor. We selectively converted neutral or anionic residues into cationic residues to obtain engineered PEDF (ePEDF). Using in vitro binding assays, we demonstrate that ePEDF had higher affinity for heparin and HA than wild-type PEDF (wtPEDF). ePEDF exhibited antiangiogenic and retinal survival bioactivities. It inhibited endothelial cell proliferation and tube formation in vitro. In an ex vivo model mimicking retinal degeneration, ePEDF protected photoreceptors from cell death. The findings suggest that protein engineering is an approach to develop active PEDF with higher ECM affinity to potentially improve its retention in the retina microenvironment and in turn make it a more efficient therapeutic drug for retinal diseases.

Aptamer photoregulation in vivo.

The in vivo application of aptamers as therapeutics could be improved by enhancing target-specific accumulation while minimizing off-target uptake. We designed a light-triggered system that permits spatiotemporal regulation of aptamer activity in vitro and in vivo. Cell binding by the aptamer was prevented by hybridizing the aptamer to a photo-labile complementary oligonucleotide. Upon irradiation at the tumor site, the aptamer was liberated, leading to prolonged intratumoral retention. The relative distribution of the aptamer to the liver and kidney was also significantly decreased, compared to that of the free aptamer.

Injectable fibroblast growth factor-2 coacervate for persistent angiogenesis.

Enhancing the maturity of the newly formed blood vessels is critical for the success of therapeutic angiogenesis. The maturation of vasculature relies on active participation of mural cells to stabilize endothelium and a basal level of relevant growth factors. We set out to design and successfully achieved robust angiogenesis using an injectable polyvalent coacervate of a polycation, heparin, and fibroblast growth factor-2 (FGF2). FGF2 was loaded into the coacervate at nearly 100% efficiency. In vitro assays demonstrated that the matrix protected FGF2 from proteolytic degradations. FGF2 released from the coacervate was more effective in the differentiation of endothelial cells and chemotaxis of pericytes than free FGF2. One injection of 500 ng of FGF2 in the coacervate elicited comprehensive angiogenesis in vivo. The number of endothelial and mural cells increased significantly, and the local tissue contained more and larger blood vessels with increased circulation. Mural cells actively participated during the whole angiogenic process: Within 7 d of the injection, pericytes were recruited to close proximity of the endothelial cells. Mature vasculature stabilized by vascular smooth muscle cells persisted till at least 4 wk. On the other hand, bolus injection of an identical amount of free FGF2 induced weak angiogenic responses. These results demonstrate the potential of polyvalent coacervate as a new controlled delivery platform.

A Biocompatible Arginine-based Polycation.

Self assembly between cations and anions is ubiquitous throughout nature. Important biological structures such as chromatin often use polyvalent assembly between a polycation and a polyaninon. Biomedical importance of synthetic polycations arises from their affinity to polyanions such as nucleic acid and heparan sulfate. However, the limited biocompatibility of synthetic polycations hampers the realization of their immense potential. By examining biocompatible cationic peptides, we hypothesize that a biocompatible polycation should be biodegradable and made from endogenous cations. We designed an arginine-based biodegradable polycation and demonstrated that it was orders of magnitude more compatible than conventional polycations in vitro and in vivo. This biocompatibility diminishes when L-arginine is substituted with D-arginine or when the biodegradable ester linker changes to a biostable ether linker. We believe this design can lead to many biocompatible polycations that can significantly advance a wide range of applications including controlled release, tissue engineering, biosensing, and medical devices.

Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo.

Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5' end of the crRNA result in enhanced serum stability. Also, extending the 5' end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5' extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo.

Advanced biomaterials and microengineering technologies to recapitulate the stepwise process of cancer metastasis.

Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.

RNA therapeutics - The potential treatment for myocardial infarction.

RNA therapeutics mainly control gene expression at the transcript level. In contrast to conventional gene therapy which solely increases production of a protein, delivered RNAs can enhance, reduce or abolish synthesis of a particular protein, which control its relevant activities in a more diverse fashion. Thus, they hold promise to treat many human diseases including myocardial infarction (MI). MI is a serious health burden that causes substantial morbidity and mortality. An unmet clinical need for treating MI is the recovery of cardiac function, which requires regeneration of the functional tissues including the vasculature, nerves, and myocardium. Several classes of RNA therapeutics have been investigated in preclinical MI models, and the results have demonstrated their benefits and encourage their future development. In this review, we summarize the common RNA therapeutic approaches and highlight their application in MI therapy.

Fabrication of biosensing surfaces using adhesive polydopamine.

Dopamine can be induced to polymerize on a variety of substrates, providing a robust and bioinspired surface coating that can be used to tune substrate surface properties and to sequester other species at the interface. We first exploit the facile nature of this surface modification procedure to generate an array of polydopamine that, in conjunction with fluorescent tags, provides the ability to detect multiple protein targets simultaneously and with great specificity. We then demonstrate the use of polydopamine as a matrix to confine gold nanoparticles at the surface of glass and graphene substrates. The nanoparticles (NPs) are used to template further gold nanoparticle growth in situ at the interface; subsequent calcination to remove the polydopamine matrix and sinter the NPs generates a highly active surface enhanced Raman scattering surface that allows for sensitive molecular detection. These varied uses in surface modification/biosensing demonstrate the utility of polydopamine as a functional surface modification for control of physical and electronic properties at the interface.

Controlled dual delivery of fibroblast growth factor-2 and Interleukin-10 by heparin-based coacervate synergistically enhances ischemic heart repair.

Myocardial infarction (MI) causes myocardial necrosis, triggers chronic inflammatory responses, and leads to pathological remodeling. Controlled delivery of a combination of angiogenic and immunoregulatory proteins may be a promising therapeutic approach for MI. We investigated the bioactivity and therapeutic potential of an injectable, heparin-based coacervate co-delivering an angiogenic factor, fibroblast growth factor-2 (FGF2), and an anti-inflammatory cytokine, Interleukin-10 (IL-10) in a spatially and temporally controlled manner. Coacervate delivery of FGF2 and IL-10 preserved their bioactivities on cardiac stromal cell proliferation in vitro. Upon intramyocardial injection into a mouse MI model, echocardiography revealed that FGF2/IL-10 coacervate treated groups showed significantly improved long-term LV contractile function and ameliorated LV dilatation. FGF2/IL-10 coacervate substantially augmented LV myocardial elasticity. Additionally, FGF2/IL-10 coacervate notably enhanced long-term revascularization, especially at the infarct area. In addition, coacervate loaded with 500 ng FGF2 and 500 ng IL-10 significantly reduced LV fibrosis, considerably preserved infarct wall thickness, and markedly inhibited chronic inflammation at the infarct area. These results indicate that FGF2/IL-10 coacervate has notably greater therapeutic potential than coacervate containing only FGF2. Overall, our data suggest therapeutically synergistic effects of FGF-2/IL-10 coacervate, particularly coacervate with FGF2 and 500 ng IL-10, for the treatment of ischemic heart disease.

Development of functional biomaterials with micro- and nanoscale technologies for tissue engineering and drug delivery applications.

Micro- and nanotechnologies have emerged as potentially effective fabrication tools for addressing the challenges faced in tissue engineering and drug delivery. The ability to control and manipulate polymeric biomaterials at the micron and nanometre scale with these fabrication techniques has allowed for the creation of controlled cellular environments, engineering of functional tissues and development of better drug delivery systems. In tissue engineering, micro- and nanotechnologies have enabled the recapitulation of the micro- and nanoscale detail of the cell's environment through controlling the surface chemistry and topography of materials, generating 3D cellular scaffolds and regulating cell-cell interactions. Furthermore, these technologies have led to advances in high-throughput screening (HTS), enabling rapid and efficient discovery of a library of materials and screening of drugs that induce cell-specific responses. In drug delivery, controlling the size and geometry of drug carriers with micro- and nanotechnologies have allowed for the modulation of parametres such as bioavailability, pharmacodynamics and cell-specific targeting. In this review, we introduce recent developments in micro- and nanoscale engineering of polymeric biomaterials, with an emphasis on lithographic techniques, and present an overview of their applications in tissue engineering, HTS and drug delivery.

The effect of a heparin-based coacervate of fibroblast growth factor-2 on scarring in the infarcted myocardium.

Effective delivery of exogenous angiogenic growth factors can provide a new therapy for ischemic diseases. However, clinical translation of growth factor therapies faces multiples challenges; the most significant one is the short half-life of the naked protein. We use heparin and a nontoxic polycation to form an injectable coacervate that protects growth factors and preserves their bioactivities. Here we report the effectiveness of fibroblast growth factor-2 (FGF2) coacervate in reducing scar burden in a mouse myocardial infarction model. The coacervate provides spatial and temporal control of the release of heparin-binding proteins. Coacervate treated animals show lower level of inflammation, fibrosis and cardiomyocyte death in the infarcted myocardium. Histological evaluation indicates that FGF2 coacervate significantly increases the number of endothelial and mural cells and results in stable capillaries and arterioles to at least 6 weeks post injection. Echocardiographic assessment shows that FGF2 coacervate promotes cardiac contractibility and inhibits ventricular dilation, suggesting that the improvement at the tissue level leads to better cardiac functions. On the contrary, identical dosage of free FGF2 shows no statistical difference from saline or vehicle control in histological or functional assessment. Overall, injection of FGF2 coacervate ameliorated the ischemic injury caused by myocardial infarction. The promising data in rodent warrant further examination of the potential of clinical translation of this technology.

Therapeutic angiogenesis: controlled delivery of angiogenic factors.

Therapeutic angiogenesis aims at treating ischemic diseases by generating new blood vessels from existing vasculature. It relies on delivery of exogenous factors to stimulate neovasculature formation. Current strategies using genes, proteins and cells have demonstrated efficacy in animal models. However, clinical translation of any of the three approaches has proved to be challenging for various reasons. Administration of angiogenic factors is generally considered safe, according to accumulated trials, and offers off-the-shelf availability. However, many hurdles must be overcome before therapeutic angiogenesis can become a true human therapy. This article will highlight protein-based therapeutic angiogenesis, concisely review recent progress and examine critical challenges. We will discuss growth factors that have been widely utilized in promoting angiogenesis and compare their targets and functions. Lastly, since bolus injection of free proteins usually result in poor outcomes, we will focus on controlled release of proteins.

Design, synthesis, and biocompatibility of an arginine-based polyester.

Polycations are very useful in biotechnology. However, most existing polycations have high toxicity that significantly limits their clinical translation. We designed poly(ethylene argininylaspartate diglyceride) (PEAD) that is based on arginine, aspartic acid, glycerol, and ethylene glycol. A set of in vitro assays demonstrated that PEAD exhibited no cytotoxicity at 1 mg/mL, which is at least 100 times higher than the widely used polycation-polyethylenimine. Subcutaneous injection of 1 mg PEAD in rats did not cause an adverse response acutely or after 4 weeks. Zeta potential measurements revealed that PEAD has high affinity to biological polyanions such as DNA and hyaluronic acid. This polycation represents a new platform of biocompatible polycations that may lead to clinical innovations in gene therapy, controlled release, tissue engineering, biosensors, and medical devices.

A [polycation:heparin] complex releases growth factors with enhanced bioactivity.

Growth factors are potent molecules that regulate cell functions including survival, self renewal, differentiation and proliferation. High-efficacy delivery of growth factors will be a powerful tool for regenerative medicine. Decades of intense research have significantly advanced the field of controlled delivery. There is, however, still a great unmet need for new methods that can improve overall efficacy of growth factor delivery. Here, we report a new growth factor delivery vehicle formed by self assembly of heparin and a biocompatible polycation, poly(ethylene argininylaspartate diglyceride) (PEAD). Of the many heparin-binding growth factors, we chose FGF-2 and NGF to demonstrate the potential of the [PEAD:heparin] delivery vehicle. The delivery vehicle incorporates both growth factors with high efficiency, controls their release, maintains the bioactivity of FGF-2 and increases the bioactivity of NGF relative to bolus delivery. [PEAD:heparin] appears to be a promising delivery matrix for many heparin-binding growth factors and may lead to efficient growth factor delivery for a variety of diseases and disabilities.

Control growth factor release using a self-assembled [polycation:heparin] complex.

The importance of growth factors has been recognized for over five decades; however their utilization in medicine has yet to be fully realized. This is because free growth factors have short half-lives in plasma, making direct injection inefficient. Many growth factors are anchored and protected by sulfated glycosaminoglycans in the body. We set out to explore the use of heparin, a well-characterized sulfated glycosaminoglycan, for the controlled release of fibroblast growth factor-2 (FGF-2). Heparin binds a multitude of growth factors and maintains their bioactivity for an extended period of time. We used a biocompatible polycation to precipitate out the [heparin:FGF-2] complex from neutral buffer to form a release matrix. We can control the release rate of FGF-2 from the resultant matrix by altering the molecular weight of the polycation. The FGF-2 released from the delivery complex maintained its bioactivity and initiated cellular responses that were at least as potent as fresh bolus FGF-2 and fresh heparin stabilized FGF-2. This new delivery platform is not limited to FGF-2 but applicable to the large family of heparin-binding growth factors.

Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice.

Plasmonic photothermal therapy (PPTT) is a minimally-invasive oncological treatment strategy in which photon energy is selectively administered and converted into heat sufficient to induce cellular hyperthermia. The present work demonstrates the feasibility of in vivo PPTT treatment of deep-tissue malignancies using easily-prepared plasmonic gold nanorods and a small, portable, inexpensive near-infrared (NIR) laser. Dramatic size decreases in squamous cell carcinoma xenografts were observed for direct (P<0.0001) and intravenous (P<0.0008) administration of pegylated gold nanorods in nu/nu mice. Inhibition of average tumor growth for both delivery methods was observed over a 13-day period, with resorption of >57% of the directly-injected tumors and 25% of the intravenously-treated tumors.

Macrophage physiological function after superparamagnetic iron oxide labeling.

Our goal was to analyze the changes in morphology and physiological function (phagocytosis, migratory capabilities, humoral and cellular response, and nitric oxide secretion) of murine macrophages after labeling with a clinically used superparamagnetic iron oxide (SPIO), ferucarbotran. In SPIO-treated macrophages, nanoparticles were taken up in the cytoplasm and accumulated in a membrane-bound organelle. Macrophage proliferation and viability were not modified after SPIO labeling. Phagocytic function decreased after labeling with only 10 microg Fe/mL SPIO, whereas other functions including migration and production of tumor necrosis factor-alpha and nitric oxide increased at the highest SPIO concentration (100 microg Fe/mL).

Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.

The purpose of this work was to evaluate the efficacy of labeling human mesenchymal stem cells (hMSCs) by ionic superparamagnetic iron oxide (SPIO) without a transfection agent and verifying its capability to be detected with clinical 1.5 T magnetic resonance (MR) at the single-cell level. Human hMSCs were incubated for 24 h with an ionic SPIO, Ferucarbotran. The labeling efficiency of hMSCs was determined by iron content measurement spectrophotometrically, and the influence of labeling on cell behavior was ascertained by examination of cell viability using the trypan blue exclusion method, cell proliferation analysis using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, mitochondrial membrane potential (MMP) change, differentiation capacity, and reactive oxygen species (ROS) production measured by dichlorofluorescein diacetate (DCFDA) fluorescent probe. Labeled hMSCs were scanned under 1.5 T MRI with three-dimensional (3D) and two-dimensional (2D) T(2)-weighted gradient echo (GRE) pulse sequences. Human hMSC labeling without transfection agent was efficient. The iron content in hMSCs was 23.4 pg Fe/cell. No significant change was found in viability, proliferation, MMP change, ROS production, or differentiation capacity. About 45.2% of the hMSCs could be detected using 1.5 T MRI at the single cell level with 3D GRE and four repetitions.

Mutations in the alpha-helix directly C-terminal to the major homology region of human immunodeficiency virus type 1 capsid protein disrupt Gag multimerization and markedly impair virus particle production.

The X-ray crystallographic structure of HIV-1 capsid protein suggests that the dimer interface of the dimerization domain is mainly formed from a putative alpha-helix structure of 14 amino acids (Gag residues 311-324) and lies directly C-terminal to the capsid major homology region. We found that a deletion mutation in the alpha-helix drastically reduces virus particle production. Alanine-scanning mutagenetic analysis indicated that substitution mutations at residues Q311, V313, K314, W316, and M317 all impair virus particle production markedly. Membrane flotation assays suggested that some mutations in the dimer interface have slight effects on the efficient binding of Gag to membranes. Indirect immunofluorescence studies revealed that mutants defective in virus production exhibit a subcellular distribution pattern similar to that of wild-type. However, velocity sedimentation analysis showed that mutations significantly impairing virus particle production were also detrimental to Gag multimerization, suggesting that the impaired virus production may be due to a defect in Gag multimerization. These results support the proposal that residues in the capsid dimer interface play a crucial role in promoting Gag multimerization, possibly by facilitating stable Gag-Gag interactions.