Felicity de Cogan

Conversion of Magnetic Impulses into Cellular Responses by Self‐Assembled Nanoparticle–Vesicle Hydrogels

Phospholipid vesicles are widely used as nano-sized drug delivery vehicles and as biomimetic model systems, where the bilayer allows fundamental biomembrane processes like ion transport, signaling, and multivalent recognition to be copied. In particular, the remotely triggered transit of stored chemicals across bilayer membranes is a key goal as it will allow vesicles to communicate with cells either in vivo or in vitro during cell culture. The latter approach should produce exciting new “smart” biomaterials, although non-invasive and non-chemical control over drug release from vesicles remains challenging. Most mammalian cells are unaffected by oscillating or permanent magnetic fields. To sensitize cells to magnetic fields, they can be labeled with magnetic nanoparticles (MNPs), an approach used to effect gene transfection with static magnetic fields or cause hyperthermia with alternating magnetic fields (AMFs). Alternatively MNPs can be used to label vesicles, which allows magnetic manipulation and AMF-triggered contents release; magnetic release is attractive as nearby cells would only be affected by the released biochemicals and not the AMF. Recently we used 10 nm Fe3O4 MNPs to crosslink 800 nm diameter phospholipid vesicles and form magnetic nanoparticle–vesicle assemblies (MNPVs). Embedding MNPVs within a hydrogel matrix added a further level of assembly, with the hydrogel fibrils acting as an artificial extracellular matrix that reinforced the vesicles, providing robust materials that responded to AMFs by releasing stored dyes. Such nanostructured and responsive self-assembled biomaterials have enormous potential in cell culture, and replacing these dyes with bioactive species should produce a new type of “smart” cell culture scaffold responsive to magnetic impulses. Herein we describe a self-assembled bionanotechnological system able to act as a “smart” biomaterial that translates non-invasive magnetic signals into cellular responses (Figure 1). An important design feature was the self-assembly of Fe3O4 nanoparticles with gel-phase vesicles, an alternative to physical incorporation that was designed to allow heat generated in the MNPs by the AMF to be efficiently transferred to the bilayers. When heated, gel-phase vesicles “melt” at a triggering temperature (Tm), an all-or-nothing event that allows complete and rapid escape of encapsulated compounds. The biotin–avidin interaction was used to link vesicles and MNPs, which improved compatibility across cell types, including myoblasts (Figure 2) and chondrocytes. It also allowed commercially available biotin lipids like N(biotinoyl)-1,2-dihexadecanoyl phosphatidylethanolamine (biotin-DHPE) to be used as vesicle crosslinkers. N-Biotinylated dopamine (1) was used to give Fe3O4 MNPs an adhesive coating. MNPs were formed by coprecipitation then sonicated with 1 in deoxygenated methanol (0.7 mm) to give 1-coated Fe3O4 nanoparticles ([1MNP]), with a coating efficiency of 50 20 % (Figure 2a). Dipalmitoyl phosphatidylcholine (DPPC) vesicles (800 nm diameter) were chosen as the nanocontainers as these bilayers have Tm 42 8C, a triggering temperature above cell culture conditions (37 8C). Vesicles with stored chemical payloads were created by extrusion of 0.2% mol/mol biotinDHPE 2 in DPPC in a solution of the compound to be encapsulated. The thermal release of encapsulated 5/6carboxyfluorescein (5/6-CF) showed these [2-DPPC] vesicles had Tm 40 8C. Addition of the avidin “glue” to a mixture of [1-MNP] and [2-DPPC] vesicles produced large magnetic Figure 1. a) Cells and nanoparticle-vesicle assemblies (MNPVs) are coimmobilized within a calcium alginate hydrogel (yellow). MNPVs are self-assembled nanocarriers composed of magnetic nanoparticles coated with N-biotinoyl dopamine 1 (1-MNP) and DPPC vesicles containing biotin-DHPE 2 (2-DPPC), which are linked together by avidin. b) Chemical messengers, such as drugs (blue), can be noninvasively released by an alternating magnetic field (AMF), and these released chemicals in turn induce responses from cultured cells.

siRNA-Mediated Knockdown of the mTOR Inhibitor RTP801 Promotes Retinal Ganglion Cell Survival and Axon Elongation by Direct and Indirect Mechanisms

PURPOSE To investigate, using in vivo and in vitro models, retinal ganglion cell (RGC) neuroprotective and axon regenerative effects and underlying mechanisms of siRTP801, a translatable small-interfering RNA (siRNA) targeting the mTOR negative regulator RTP801. METHODS Adult rats underwent optic nerve (ON) crush (ONC) followed by intravitreal siRTP801 or control siRNA (siEGFP) every 8 days, with Brn3a+ RGC survival, GFAP+ reactive gliosis, and GAP43+ regenerating axons analyzed immunohistochemically 24 days after injury. Retinal cultures, prepared from uninjured animals or 5 days after ONC to activate retinal glia, were treated with siRTP801/controls in the presence/absence of rapamycin and subsequently assessed for RGC survival and neurite outgrowth, RTP801 expression, glial responses, and mTOR activity. Conditioned medium was analyzed for neurotrophin titers by ELISA. RESULTS Intravitreal siRTP801 enabled 82% RGC survival compared to 45% with siEGFP 24 days after ONC, correlated with greater GAP43+ axon regeneration at 400 to 1200 μm beyond the ONC site, and potentiated the reactive GFAP+ Müller glial response. In culture, siRTP801 had a direct RGC neuroprotective effect, but required GFAP+ activated glia to stimulate neurite elongation. The siRTP801-induced neuroprotection was significantly reduced, but not abolished, by rapamycin. The siRTP801 potentiated the production and release of neurotrophins NGF, NT-3, and BDNF, and prevented downregulation of RGC mTOR activity. CONCLUSIONS The RTP801 knockdown promoted RGC survival and axon elongation after ONC, without increasing de novo regenerative sprouting. The neuroprotection was predominantly direct, with mTORC1-dependent and -independent components. Enhanced neurite/axon elongation by siRTP801 required the presence of activated retinal glia and was mediated by potentiated secretion of neurotrophic factors.

Peptide aptamers: Novel coatings for orthopaedic implants

Current processes for coating titanium implants with ceramics involve very high energy techniques with associated high cost and disadvantages such as heterogeneity of the coatings, phase transformations and inability to coat complex structures. In order to address the above problems, we propose a biomimetic hydroxyapatite coating process with the use of peptides that can bind both on titanium surfaces and hydroxyapatite. The peptides enabled homogeneous coating of a titanium surface with hydroxyapatite. The hydroxyapatite-peptide sandwich coating showed no adverse effects on cell number or collagen deposition. This makes the sandwich coated titanium a good candidate for titanium implants used in orthopaedics and dentistry.

Spatially controlled apoptosis induced by released nickel(ii) within a magnetically responsive nanostructured biomaterial

Magnetically patterned and responsive biomaterials have been shown to produce spatially controlled cell death in response to a magnetic signal. The responsive elements in these nano-structured biomaterials are magnetic nanoparticle-vesicle assemblies (MNPVs), which are thermally sensitive vesicles crosslinked by magnetic nanoparticles. MNPVs are nano-sized drug delivery platforms that are responsive to magnetic fields in two ways: they can be spatially manipulated by static magnetic fields, and upon exposure to an alternating magnetic field (AMF) they can release chemical messengers stored within the vesicles. Magnetically initiated release of nickel(II) from MNPVs immobilised in an alginate hydrogel was used to produce remotely triggered and spatially controlled apoptosis of fibroblasts cultured in the hydrogel. The ability to manipulate MNPVs with static magnetic fields was used to immobilise the MNPVs in only one region of the biomaterial; subsequent AMF-induced release of nickel(II) caused a wave of cellular apoptosis through the biomaterial as the nickel(II) slowly diffused through the hydrogel.

Decorin Reduces Intraocular Pressure and Retinal Ganglion Cell Loss in Rodents Through Fibrolysis of the Scarred Trabecular Meshwork

PURPOSE To investigate whether Decorin, a matrikine that regulates extracellular matrix (ECM) deposition, can reverse established trabecular meshwork (TM) fibrosis, lower IOP, and reduce progressive retinal ganglion cell (RGC) death in a novel rodent model of TM fibrosis. METHODS Adult rats had intracameral (IC) injections of human recombinant (hr) TGF-β over 30 days (30 d; to induce TM fibrosis, raise IOP, and initiate RGC death by 17 d) or PBS (controls) and visually evoked potentials (VEP) were measured at 30 d to evaluate resultant visual pathway dysfunction. In some animals TGF-β injections were stopped at 17 d when TM fibrosis and IOP were consistently raised and either hrDecorin or PBS IC injections were administered between 21 d and 30 d. Intraocular pressure was measured biweekly and eyes were processed for immunohistochemical analysis of ECM deposition to assess TM fibrosis and levels of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) to assess fibrolysis. The effect of hrDecorin treatment on RGC survival was also assessed. RESULTS Transforming growth factor-β injections caused sustained increases in ECM deposition in the TM and raised IOP by 17 d, responses that were associated with 42% RGC loss and a significant decrease in VEP amplitude measured at 30 d. Decorin treatment from 17 d reduced TGF-β-induced TM fibrosis, increased levels of MMP2 and MMP9 and lowered TIMP2 levels, and lowered IOP, preventing progressive RGC loss. CONCLUSIONS Human recombinant Decorin reversed established TM fibrosis and lowered IOP, thereby rescuing RGC from progressive death. These data provide evidence for the candidacy of hrDecorin as a treatment for open-angle glaucoma.

Adhesive interactions between cells and biotinylated phospholipid vesicles in alginate: towards new responsive biomaterials.

Creating tissue-mimetic biomaterials able to deliver bioactive compounds after receipt of a remote and non-invasive trigger has so far proved to be challenging. The possible applications of such “smart” biomaterials are vast, ranging from subcutaneous drug delivery to tissue engineering. Self-assembled phospholipid vesicles (liposomes) have the ability to deliver both hydrophilic and hydrophobic drugs, and controlling interactions between functionalized vesicles and cells within biomaterials is an important step for targeted drug delivery to cells. We report an investigation of the interactions between thermally-sensitive and biotin-coated dipalmitoyl phosphatidylcholine vesicles and 3T3 fibroblast cells. The stability of these vesicles under physiological conditions was assessed and their interaction with the cell membranes of fibroblasts in media and alginate/fibronectin mixtures was studied. Stable vesicle-cell aggregates were formed in fluid matrices, and could be a model system for improving the delivery of remotely released drugs within vesicle-containing biomaterials.

Topical Delivery of Anti-VEGF Drugs to the Ocular Posterior Segment Using Cell-Penetrating Peptides

Purpose To evaluate the efficacy of anti-VEGF agents for treating choroidal neovascularization (CNV) when delivered topically using novel cell-penetrating peptides (CPPs) compared with delivery by intravitreal (ivit) injection. Methods CPP toxicity was investigated in cell cultures. Ivit concentrations of ranibizumab and bevacizumab after topical administration were measured using ELISA. The biological efficacy of topical anti-VEGF + CPP complexes was compared with ivit anti-VEGF injections using an established model of CNV. Results CPPs were nontoxic in vitro. In vivo, after topical eye drop delivery, CPPs were present in the rat anterior chamber within 6 minutes. A single application of CPP + bevacizumab eye drop delivered clinically relevant concentrations of bevacizumab to the posterior chamber of the rat eye in vivo. Similarly, clinically relevant levels of CPP + ranibizumab and CPP + bevacizumab were detected in the porcine vitreous and retina ex vivo. In an established model of CNV, mice treated with either a single ivit injection of anti-VEGF, twice daily CPP + anti-VEGF eye drops or daily dexamethasone gavage for 10 days all had significantly reduced areas of CNV when compared with lasered eyes without treatment. Conclusions CPPs are nontoxic to ocular cells and can be used to deliver therapeutically relevant doses of ranibizumab and bevacizumab by eye drop to the posterior segment of mouse, rat, and pig eyes. The CPP + anti-VEGF drug complexes were cleared from the retina within 24 hours, suggesting a daily eye drop dosing regimen. Daily, topically delivered anti-VEGF with CPP was as efficacious as a single ivit injection of anti-VEGF in reducing areas of CNV in vivo.

Thermosensitive hydrogel as an in situ gelling antimicrobial ocular dressing

Microbial keratitis is a severe ocular condition and one of the most prevalent causes of corneal scarring and associated blindness worldwide. Risk factors include contact lens use, ocular trauma, ocular surface disease and immunosuppression. Initial clinical management mandates intensive (hourly or more frequent) topical administration of broad spectrum antimicrobial therapy for at least 48h, which may require hospital admission, followed by tailored therapy based on microbiological investigation and the institution of strategies to reduce inflammation and promote healing. In this work we report an ocular wound dressing which can encapsulate and give sustained release of different antibiotics. The use of this dressing would allow patients to have eye drops on a 4 hourly basis, thereby facilitating treatment compliance and reducing hospital admissions.

Antimicrobial peptide coatings for hydroxyapatite: electrostatic and covalent attachment of antimicrobial peptides to surfaces

The interface between implanted devices and their host tissue is complex and is often optimized for maximal integration and cell adhesion. However, this also gives a surface suitable for bacterial colonization. We have developed a novel method of modifying the surface at the material–tissue interface with an antimicrobial peptide (AMP) coating to allow cell attachment while inhibiting bacterial colonization. The technology reported here is a dual AMP coating. The dual coating consists of AMPs covalently bonded to the hydroxyapatite surface, followed by deposition of electrostatically bound AMPs. The dual approach gives an efficacious coating which is stable for over 12 months and can prevent colonization of the surface by both Gram-positive and Gram-negative bacteria.