Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy

We recently reported that the Ins2Akita mouse is a good model for late-onset diabetic retinopathy. Here, we investigated the effect of miR200-b, a potential anti-angiogenic factor, on VEGF receptor 2 (VEGFR-2) expression and to determine the underlying angiogenic response in mouse endothelial cells, and in retinas from aged Ins2Akita mice. MiR200-b and its native flanking sequences were amplified and cloned into a pCAG-eGFP vector directed by the ubiquitous CAG promoter (namely pCAG-miR200-b-IRES-eGFP). The plasmid was compacted by CK30PEG10K into DNA nanoparticles (NPs) for in vivo delivery. Murine endothelial cell line, SVEC4-10, was first transfected with the plasmid. The mRNA levels of VEGF and VEGFR-2 were quantified by qRT-PCR and showed significant reduction in message expression compared with lipofectamine-transfected cells. Transfection of miR200-b suppressed the migration of SVEC4-10 cells. There was a significant inverse correlation between the level of expression of miR200-b and VEGFR-2. Intravitreal injection of miR200-b DNA NPs significantly reduced protein levels of VEGFR-2 as revealed by western blot and markedly suppressed angiogenesis as evaluated by fundus imaging in aged Ins2Akita mice even after 3 months of post-injection. These findings suggest that NP-mediated miR200-b delivery has negatively regulated VEGFR-2 expression in vivo

A “Trojan Horse” Strategy: The Preparation of Bile Acid-Modifying Irinotecan Hydrochloride Nanoliposomes for Liver-Targeted Anticancer Drug Delivery System Study

The bile acid transport system is a natural physiological cycling process between the liver and the small intestine, occurring approximately 6–15 times during the day. There are various bile acid transporter proteins on hepatocytes that specifically recognize bile acids for transport. Therefore, in this paper, a novel liposome, cholic acid-modified irinotecan hydrochloride liposomes (named CA-CPT-11-Lip), was prepared based on the “Trojan horse” strategy. The liposomes preparation process was optimized, and some important quality indicators were investigated. The distribution of irinotecan hydrochloride in mice was then analyzed by high-performance liquid chromatography (HPLC), and the toxicity of liposomes to hepatocellular carcinoma cells (HepG-2) was evaluated in vitro. As a result, CA-CPT-11-Lip was successfully prepared. It was spherical with a particle size of 154.16 ± 4.92 nm, and the drug loading and encapsulation efficiency were 3.72 ± 0.04% and 82.04 ± 1.38%, respectively. Compared with the conventional liposomes (without cholic acid modification, named CPT-11-Lip), CA-CPT-11-Lip had a smaller particle size and higher encapsulation efficiency, and the drug accumulation in the liver was more efficient, enhancing the anti-hepatocellular carcinoma activity of irinotecan hydrochloride. The novel nanoliposome modified by cholic acid may help to expand the application of irinotecan hydrochloride in the treatment of hepatocellular carcinoma and construct the drug delivery system mode of drug liver targeting

A “Trojan Horse” Strategy: The Preparation of Bile Acid-Modifying Irinotecan Hydrochloride Nanoliposomes for Liver-Targeted Anticancer Drug Delivery System Study

The bile acid transport system is a natural physiological cycling process between the liver and the small intestine, occurring approximately 6–15 times during the day. There are various bile acid transporter proteins on hepatocytes that specifically recognize bile acids for transport. Therefore, in this paper, a novel liposome, cholic acid-modified irinotecan hydrochloride liposomes (named CA-CPT-11-Lip), was prepared based on the “Trojan horse” strategy. The liposomes preparation process was optimized, and some important quality indicators were investigated. The distribution of irinotecan hydrochloride in mice was then analyzed by high-performance liquid chromatography (HPLC), and the toxicity of liposomes to hepatocellular carcinoma cells (HepG-2) was evaluated in vitro. As a result, CA-CPT-11-Lip was successfully prepared. It was spherical with a particle size of 154.16 ± 4.92 nm, and the drug loading and encapsulation efficiency were 3.72 ± 0.04% and 82.04 ± 1.38%, respectively. Compared with the conventional liposomes (without cholic acid modification, named CPT-11-Lip), CA-CPT-11-Lip had a smaller particle size and higher encapsulation efficiency, and the drug accumulation in the liver was more efficient, enhancing the anti-hepatocellular carcinoma activity of irinotecan hydrochloride. The novel nanoliposome modified by cholic acid may help to expand the application of irinotecan hydrochloride in the treatment of hepatocellular carcinoma and construct the drug delivery system mode of drug liver targeting

Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors

Subretinal delivery of AAVs and NPs efficiently transduces retinal tissues.

<p>Balb/C mice were subretinally injected at P30 with Naked-CBA-GFP, Naked-MOP-GFP, NP-CBA-GFP, NP-MOP-GFP (6.9¹¹ vg), AAV2-CBA-GFP, or AAV5-MOP-GFP (10⁹ vg). Native GFP fluorescence was imaged using a spinning disk confocal microscope at PI-2 (<b>a</b>), PI-14, PI-30 (<b>b</b>), and PI-90 (<b>c</b>). Negative control images are shown in (<b>d</b>). Scale bars: 40 µm. <b>e</b>. Sections were labeled with antibodies against rod opsin (purple) and S-opsin (red), and nuclei were counterlabeled with DAPI. Green is GFP native fluorescence. Arrows show cone photoreceptors which express GFP, while arrowheads highlight cones which do not express GFP. N = 3 eyes/group. Scale bar: 10 µm. Shown are representative single planes from confocal stacks. To control for normal retinal autofluorescence, images were captured at equivalent exposure times from control and experimental eyes. OS: outer segment, IS: inner segment, ONL: outer nuclear layer, INL: inner nuclear layer. N = 3–5 eyes/cohort.</p

Subretinal injection of AAVs but not NPs leads to GFP expression in the brain.

<p>P30 Balb/C mice were subretinally injected bilaterally with NP-CBA-GFP, NP-MOP-GFP (6.9¹¹ vg), AAV2-CBA-GFP, or AAV5-MOP-GFP (10⁹ vg). <b>a.</b> Native GFP fluorescence in the optic nerve (ON) was assessed by examination of central retinal cryosections. <b>b.</b> Whole brain schematic showing approximate regions where slices shown in (<b>c</b>), are captured. <b>c.</b> Transverse cryosections of whole brain were prepared for confocal microscopy at PI-90 days. Shown are representative low magnification (top row) and higher magnification (bottom row-magnification of box) images of native GFP fluorescence in the visual tract in animals injected with AAV2-CBA-GFP. <b>d.</b> GFP vector DNA was amplified from genomic DNA harvested from the eye, ON, optic chiasm, lateral geniculate nucleus (LGN), and visual cortex (VC). Shown are representative agarose gels from samples analyzed at PI-30 and PI-60 days. N = 8 (eye, ON, LGN, VC) and 4 (chiasm). cp: cerebral crus; InG: layers of superior colliculus; Op: optic nerve layer of the superior colliculus; opt: optic tract; ox: optic chasim; sox: supraoptic decussation; so: supraoptic. Scale bars <b>b.</b> 40 µm, <b>d.</b> 600 µm (top) and 160 µm (bottom). N values for <b>a, b</b> and <b>d</b> can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052189#pone-0052189-t001" target="_blank"><b>Table 1</b></a>.</p

GFP transduced cells are distributed throughout the retina in AAV and NP-treated animals and persist for up to one year.

<p>Balb/C mice were subretinally injected at P30 with Naked-CBA-GFP, Naked-MOP-GFP, NP-CBA-GFP, NP-MOP-GFP (6.9¹¹ vg), AAV2-CBA-GFP, or AAV5-MOP-GFP (10⁹ vg). GFP distribution was examined <i>in vivo</i> by brightfield/GFP fundus imaging at PI-2 (a-arrows show regions of early expression), PI-30 (b-top row), and PI-360 (d). Distribution was also assessed by capturing images of native GFP fluorescence in entire sections cut through the optic nerve at PI-30 (b-bottom row), and PI-90 (c). Arrows in b, c show the approximate region of injection. Scale bar, 500 µm. N-nasal, T-temporal. N = 3–5 eyes/cohort.</p

GFP expression is restricted to the retina of NP-injected animals but is found in the brain of AAV-treated animals.

<p>GFP expression by native fluorescence and vector DNA detection by qPCR after bilateral subretinal injection of AAVs and NPs. Values expressed as (# expressing/# examined).</p

NP- and AAV-mediated gene expression persisted for up to four months.

<p>P30 Balb/C mice were subretinally injected with Naked-CBA-GFP, Naked-MOP-GFP, NP-CBA-GFP, NP-MOP-GFP (6.9¹¹ vg), AAV2-CBA-GFP, or AAV5-MOP-GFP (10⁹ vg). <b>a.</b> GFP mRNA levels (normalized to β-actin ) in whole eyes were assessed by qRT-PCR at the indicated timepoints. <b>B.</b> Shown are representative western blots probed with antibodies against GFP/actin to assess GFP protein expression in the retina at various timepoints PI. <b>c.</b> Densitometric quantification of western blots represented in (<b>b</b>). GFP band densities were quantitated, normalized to β-actin, and expressed as a fraction of that found in GPI-GFP transgenic mice (used as a positive control in every blot). <b>a, c.</b> N = 6/cohort, shown are means ± SEM *p<0.01, **p<0.001 for comparsion between AAV and NP, by two-way ANOVA with Bonferroni’s post-hoc test.</p