Development of Long-Circulating Zwitterionic Cross-Linked Micelles for Active-Targeted Drug Delivery

Blood stability, active targeting, and controlled drug release are the most important features to design desirable drug carriers. Here, we demonstrate a zwitterionic biodegradable cross-linked micelle based on a penta-block copolymer, which utilizes poly­(carboxybetaine methacrylate) as hydrophilic segment, poly­(ε-caprolactone) as biodegradable hydrophobic segment, poly­(<i>S</i>-2-hydroxyethyl-<i>O</i>-ethyl dithiocarbonate methacrylate) (PSODMA) block as thiol protecting segment for cross-linking, and cyclic Arg-Gly-Asp-d-Tyr-Lys [c­(RGDyK)] as targeting ligand. As a result, this micelle possessed excellent colloidal stability at high dilution and in 50% fetal bovine serum. In vitro drug release experiment showed no burst release under physiological conditions but accelerated drug release in mimicking tumor tissue environment. In vivo tests showed that the drug-loaded micelles had prolonged half-life in bloodstream, enhanced therapeutic efficiency, and reduced cardiac toxicity and biotoxicity compared with free drug formulation. Taken together, the reported c­(RGDyK)-modified zwitterionic interfacially cross-linked micelle has emerged as an appealing platform for cancer therapy

Development of Zwitterionic Polypeptide Nanoformulation with High Doxorubicin Loading Content for Targeted Drug Delivery

Much attention has been drawn to targeted nanodrug delivery systems due to their high therapeutic efficacy in cancer treatment. In this work, doxorubicin (DOX) was incorporated into a zwitterionic arginyl–glycyl–aspartic acid (RGD)-conjugated polypeptide by an emulsion solvent evaporation technique with high drug loading content (45%) and high drug loading efficiency (95%). This zwitterionic nanoformulation showed excellent colloidal stability at high dilution and in serum. The pH-induced disintegration and enzyme-induced degradation of the nanoformulation were confirmed by dynamic light scattering and gel permeation chromatography. Efficient internalization of DOX in the cells and high antitumor activity in vitro was observed. Compared with the free drug, this nanoformulation showed higher accumulation in tumor and lower systemic toxicity in vivo. The DOX-loaded zwitterionic RGD-conjugated polypeptide vesicles show potential application for targeted drug delivery in the clinic

Metabolomics analysis of <i>Comtd1</i>-KO and wild-type B16F10 cells.

Principal component analysis (PCA) and Partial Least-Squares Discriminant Analysis (PLS-DA) with model validation results. PCA is an unsupervised multivariate analysis, which provides an unbiased overview of the metabolite features due to unawareness of the two groups compared (WT and KO in this case). PLS-DA is a multivariate analysis which considers data from the two groups and select the most discriminating metabolites that separates the two groups. This is the reason why PLS-DA show a better separation between WT and KO groups. (A) PCA in MS positive mode detection. (B) PLS-DA in MS positive mode detection. (C) PCA in MS negative mode detection. (D) PLS-DA in MS negative mode detection. (E) Significantly altered pathways impacted by Comtd1 knockout. (PDF)</p

HA-tagged COMTD1 localizes to mitochondria in immortalized mouse melanocytes.

(A-D) Immortalized melan-Ink4a cells from Ink4a-deficient C57BL/6J mice were transiently transfected to express COMTD1 fused with the HA11 epitope at either the N-terminus (HA-COMTD1; A, C) or C-terminus (COMTD1-HA; B, D). Two days later, cells were fixed and analyzed by bright field (BF) and immunofluorescence microscopy for HA and either the mitochondrial resident protein MAVS (A, B) or the ER resident protein calnexin (CNX; C, D). Individual images of labelled cells or the bright field image are shown in addition to an overlay of HA (green) with MAVS (red; HA/ MAVS), CNX (red; HA/ CNX), or the pseudocolored bright field image (magenta; HA/BF). Insets show a 5-fold magnified image of the boxed region to emphasize overlap or lack thereof. Main scale bar, 10 μm; inset scale bar, 2 μm. (E) Quantification of the degree of overlap of COMTD1-HA or HA-COMTD1, as indicated, with markers of the ER (CNX; N = 29 for COMTD1-HA, N = 17 for HA-COMTD1), mitochondria (MAVS; N = 25 for COMTD1-HA, N = 16 for HA-COMTD1), mature melanosomes (TYRP1; N = 16), immature melanosomes (PMEL; N = 17), late endosomes/ lysosomes (LAMP2; N = 15), or early endosomes (STX13; N = 21). Data from 4–5 individual experiments are presented as a box and whiskers plot in which the area of overlap is shown relative to the total area occupied by HA (e.g., CNX vs. HA) or by the indicated marker (e.g., HA vs. CNX). See S2 Fig for examples of the data for TYRP1, PMEL, LAMP2 and STX13. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s tests for multiple comparisons; ****, P < 0.0001.</p

Chemical characterization of feather melanin.

(A) depicts levels of total melanin in wild-type birds (R+ and R-) and in IG birds analyzed by Soluene-350 solubilization. (B) depicts A650/A500 ratios analyzed by Soluene-350 solubilization. (C) depicts eumelanin (EM), benzothiazine-pheomelanin (BT-PM), and benzothiazole-pheomelanin (BZ-PM) analyzed as PTCA, 4-AHP, and TTCA, respectively. Feather samples were obtained from neck regions from 3 males and 3 females. Results are shown with the means ± SEM of 6 birds. *** and ** indicate P P = 0.0001–0.01 between R- and IG birds and between R- and R+ birds (Student’s t test).</p

Primer sequences used in this paper.

The biochemical pathway regulating the synthesis of yellow/red pheomelanin is less well characterized than the synthesis of black/brown eumelanin. Inhibitor of gold (IG phenotype) is a plumage colour variant in chicken that provides an opportunity to further explore this pathway since the recessive allele (IG) at this locus is associated with a defect in the production of pheomelanin. IG/IG homozygotes display a marked dilution of red pheomelanin pigmentation, whilst black pigmentation (eumelanin) is only slightly affected. Here we show that a 2-base pair insertion (frame-shift mutation) in the 5th exon of the Catechol-O-methyltransferase containing domain 1 gene (COMTD1), expected to cause a complete or partial loss-of-function of the COMTD1 enzyme, shows complete concordance with the IG phenotype within and across breeds. We show that the COMTD1 protein is localized to mitochondria in pigment cells. Knockout of Comtd1 in a mouse melanocytic cell line results in a reduction in pheomelanin metabolites and significant alterations in metabolites of glutamate/glutathione, riboflavin, and the tricarboxylic acid cycle. Furthermore, COMTD1 overexpression enhanced cellular proliferation following chemical-induced transfection, a potential inducer of oxidative stress. These observations suggest that COMTD1 plays a protective role for melanocytes against oxidative stress and that this supports their ability to produce pheomelanin.</div

Genotype distribution of the 2-bp insertion in <i>COMTD1</i> associated with the Inhibitor of Gold (IG) phenotype in different populations sorted by phenotype.

Genotype distribution of the 2-bp insertion in COMTD1 associated with the Inhibitor of Gold (IG) phenotype in different populations sorted by phenotype.</p

Sequence variants (non-reference alleles) in the coding region of <i>LRMDA</i>, <i>ZNF503</i>, and <i>COMTD1</i> detected by sequencing one homozygous <i>IG/IG</i> bird and the corresponding allele frequencies among non-IG <i>(N/-</i>) birds.

Sequence variants (non-reference alleles) in the coding region of LRMDA, ZNF503, and COMTD1 detected by sequencing one homozygous IG/IG bird and the corresponding allele frequencies among non-IG (N/-) birds.</p

Illustration of plumage phenotypes associated with different genotypes at the <i>Inhibitor of gold</i> locus in chicken on different genetic backgrounds.

The birds in (A) and (B) carry the bottom recessive wheaten allele (e) at the MC1R locus and shows red pheomelanin-based pigmentation. The birds in (C) and (D) carry the brown allele (eb) at the same locus that allows expression of both eumelanin and pheomelanin and IG dilution is apparent as regards pheomelanin pigmentation. (A) and (B) depict F2 birds from the mapping pedigree with the wild-type phenotype or the recessive IG phenotype (IG/IG), respectively. (C) and (D) depict two IG/IG birds from the Lemon Millefleur Sabelpoot (Fig 1C) and Sebright-Lemon (Fig 1D) breeds, respectively. Photo by Michèle Tixier-Boichard (A and B) and C and D were taken by Nicolas Bruneau, INRAE (C and D).</p

Genotype data at SNP positions of the <i>IG</i> locus used for identical-by-descent mapping.

Genotype data at SNP positions of the IG locus used for identical-by-descent mapping.</p