19 research outputs found

    Sheddable Coatings for Long-Circulating Nanoparticles

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    Nanoparticles, such as liposomes, polymeric micelles, lipoplexes and polyplexes are frequently studied as targeted drug carrier systems. The ability of these particles to circulate in the bloodstream for a prolonged period of time is often a prerequisite for successful targeted delivery. To achieve this, hydrophilic ‘stealth’ polymers, such as poly(ethylene glycol) (PEG), are used as coating materials. Such polymers shield the particle surface and thereby reduce opsonization by blood proteins and uptake by macrophages of the mononuclear phagocyte system. Yet, after localizing in the pathological site, nanoparticles should deliver their contents in an efficient manner to achieve a sufficient therapeutic response. The polymer coating, however, may hinder drug release and target cell interaction and can therefore be an obstacle in the realization of the therapeutic response. Attempts have been made to enhance the therapeutic efficacy of sterically stabilized nanoparticles by means of shedding, i.e. a loss of the coating after arrival at the target site. Such an ‘unmasking’ process may facilitate drug release and/or target cell interaction processes. This review presents an overview of the literature regarding different shedding strategies that have been investigated for the preparation of sterically stabilized nanoparticulates. Detach mechanisms and stimuli that have been used are described

    IN VIVO DELIVERY OF NAKED AND LIPID-COMPLEXED ANTISENSE OLIGOS IN MDX MICE: EFFECTS ON SKELETAL AND CARDIAC MUSCLE

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    Antisense-mediated exon skipping holds great potential for the treatment of DMD. In mdx mice, functional recovery of skeletal muscle has been reported upon systemic delivery of \u201cnaked\u201d oligonucleotides or viral vectors encoding for antisense snRNAs. However, only one study achieved dystrophin restoration in cardiac muscle (using an adeno-associated vector). Here we report the in vivo delivery of morpholino oligos in aged mdx mice, both in skeletal muscle, via intra-arterial injection, and in cardiac muscle, via intramuscular injection. Intra-arterial delivery yielded levels of dystrophin restoration comparable to those reported in the literature with the intra-venous approach, but with smaller amounts of oligonucleotides. Intra-cardiac injections, on the other hand, showed that the level and duration of the skipping effect found in cardiac muscle were greatly decreased compared to skeletal muscle. This latter finding provides the first direct evidence that antisense-mediated dystrophin restoration in cardiac muscle might suffers from limitations that do not exist in skeletal muscle. All data published so far have indicated that systemic delivery via the vasculature requires large amount of naked oligos to achieve therapeutically significant results. Here we also report that the use of lipid carriers has the potential to greatly improve the delivery efficiency; in particular, we found that the use of lipid-encapsulated oligo RNA allowed to detect dystrophin re-expression with a single dose of ~40 \ub5g of oligos per adult mdx mouse. Importantly, dystrophin restoration could be seen not only in skeletal and but also (albeit to a smaller extent) in cardiac muscle

    Systemic delivery of therapeutic nucleic acids into skeletal muscle by means of lipid-based vectors

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    Introduction : Lipid-based vectors are considered a promising tool when a systemic delivery is desirable. Although not as efficient as viral vectors, lipoplexes present some major advantages, as they are non immunogenic, non mutagenic, as well as easy and cheap to prepare. We had previously shown that skeletal muscles are poorly accessible to lipoplexes via intra-venous administration, whereas local intra-arterial delivery yielded good preliminary results. Objectives : Optimization of a lipid-based, systemic gene delivery system capable of introducing therapeutic nucleic acids into skeletal muscle. Methods : We use lipid-based vehicles containing the cationic lipid DODAC, formulated either as conventional or encapsulated PEG-ylated lipoplexes. Results : We injected lipoplexes in the femoral artery of adult rats in which muscle regeneration was chemically induced in the TA muscles. Using an encapsulated formulation (SPLP, developed by Protiva Bioteherapeutics) we obtained up to 10% of GFP+ve fibers after a single injection. High transfection levels were maintained for at least three weeks. At the same time, we showed that SPLPs only induced a slight delay in the process of muscle regeneration and did not lead to any germ-line transmission of the transgene. More recently we began to apply our delivery protocol to the development of an exon-skipping approach for the correction of mutations in the dystrophin gene. In particular, we are using DODACbased lipoplexes to deliver an exon 23-specific morpholino AO into the muscles of mdx mice. Our initial data showed the production of a significant amount (i.e., as abundant as the wild type or higher) of the desired skipped mRNA in the leg muscle groups of the injected limbs. The effect of the treatment on dystrophin production is presently being assessed. Conclusions : Our data indicate that both encapsulated and conventional lipoplexes have the potential to be used for the development of therapeutic protocols for muscle diseases

    Real Time Measurement of PEG Shedding from Lipid Nanoparticles in Serum via NMR Spectroscopy

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    Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems
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