Enhance Cancer Cell Recognition and Overcome Drug Resistance Using Hyaluronic Acid and α‑Tocopheryl Succinate Based Multifunctional Nanoparticles

Multidrug resistance (MDR) presents a clinical obstacle to cancer chemotherapy. The main purpose of this study was to evaluate the potential of a hyaluronic acid (HA) and α-tocopheryl succinate (α-TOS) based nanoparticle to enhance cancer cell recognition and overcome MDR, and to explore the underlying mechanisms. A multifunctional nanoparticle, HTTP-50 NP, consisted of HA-α-TOS (HT) conjugate and d-α-tocopheryl polyethylene glycol succinate (TPGS) with docetaxel loaded in its hydrophobic core. The promoted tumor cell recognition and accumulation, cytotoxicity, and mitochondria-specific apoptotic pathways for the HTTP-50 NP were confirmed in MCF-7/Adr cells (P-gp-overexpressing cancer model), indicating that the formulated DTX and the conjugated α-TOS in the HTTP-50 NP could synergistically circumvent the acquired and intrinsic MDR in MCF-7/Adr cells. <i>In vivo</i> investigation on the MCF-7/Adr xenografted nude mice models confirmed that HTTP-50 NP possessed much higher tumor tissue accumulation and exhibited pronouncedly enhanced antiresistance tumor efficacy with reduced systemic toxicity compared with HTTP-0 NP and Taxotere. The mechanisms of the multifunctional HTTP-50 NP to overcome MDR and enhance antiresistance efficacy may be contributed by CD44 receptor-targeted delivery and P-gp efflux inhibition, and meanwhile to maximize antitumor efficacy by synergism of DTX and mitocan of α-TOS killing tumor cells

DataSheet1_Two novel variants in CEP152 caused Seckel syndrome 5 in a Chinese family.doc

Background: Seckel syndrome (SCKL) is a rare autosomal recessive inherited disorder, which is mainly characterized by intrauterine and postnatal growth restrictions, microcephaly, intellectual disability, and a typical “bird-head” facial appearance. Here, we aimed to identify the genetic etiology of a family with suspected SCKL.Methods: This study enrolled a Chinese family suspected of SCKL with their detailed family history and clinical data. We performed karyotype analysis, copy number variation sequencing (CNV-seq), and trio whole-exome sequencing (WES) to explore the genetic etiology in the proband. Furthermore, the quantitative real-time polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) were conducted to confirm the pathogenicity of novel variants.Results: The karyotype analysis and CNV-seq were normal in the proband. Two novel variants in CEP152, c.1060C>T (p.Arg354*) and c.1414-14A>G, were identified in the proband through trio-WES. The qPCR results showed that the total CEP152 mRNA expression levels were significantly reduced in c.1060C>T (p.Arg354*) and c.1414-14A>G compared with healthy control individuals. Moreover, aberrant skipping of exon 12 due to the non-canonical splice-site variant was revealed by RT-PCR and Sanger sequencing.Conclusion: Our findings expanded pathogenic variant spectra in SCKL and offered new insights into the pathogenicity of a non-classical splice-site variant in CEP152, which provided additional information for helping the family improve pregnancy plans in the future.</p

Additional file 1: Figure S1. of Three novel mutations of STK11 gene in Chinese patients with Peutz–Jeghers syndrome

Polyphen-2 reports for the pathogenicity of the mutant amino acid residues in STK11. The amino acid substitution p.Ala241Pro was valued “PROBABLY DAMAGING” with the score 0.999 (HumDiv) and 0.971 (HumVar). (TIF 95 kb

Teratoma formation in immunodeficiency mice by targeted cells.

<p>H&E staining of teratomas was performed. Derivatives of all three germ layers were observed in the endoderm: (<b>a</b>) gallbladder, (<b>b</b>) intestinal-like epithelium, and (<b>c</b>) respiratory epithelium; in the mesoderm, (<b>d</b>) cartilage and (<b>e</b>) muscle; and in the ectoderm, (<b>f</b>) squamous epithelium. Bar  = 200 µm.</p

Expression of the transgene in targeted cells.

<p>(<b>a</b>) RT-PCR analysis of F9expression in targeted clones. The expected product was amplified from all analyzed homologous recombinants after long-term culture (more than 30 passages), while no transcript was detected in wild-type H9 cells. (<b>b</b>) Western blot of the clone’s lysate using an antibody anti human F9 protein. (<b>c</b>) ELISA analysis of supernatants from recombinant culture. No F9 secretion was detected from wild-type H9 cells. The targeted clones secreted the protein at different levels.</p

Gene targeting of the rDNA locus in hESCs.

<p>(<b>a</b>) For hESC transfection, the efficiency was determined by transient nucleofection of H9 single cells by pmaxGFP. (<b>b</b>) Phase-contrast image of a resistant clone just before picking up. (<b>c</b>) After two weeks of drug selection, a portion of resistant clones were picked up and the remaining clones were fixed and stained with Giemsa stain. Clones with diameters of ≥2 millimeters were considered resistant. (<b>d</b>) PCR result using P1 and P2, and DNA sequencing of the PCR product. (<b>e</b>) The targeted clones showed a normal karyotype (46, XX). (<b>f–g</b>) Southern blot analysis showed a 7.1 kb band for targeted clones using a 5′ probe. When using a 3′ probe corresponding to the second exon of <i>F9</i> gene, both a wild type 4.3 kb fragment and an integrated 10.3 kb fragment will be detected. M, marker. Scale bar  = 200 µm (a–b).</p

<i>In vitro</i> differentiation of targeted clones.

<p>Immunostaining images show cells derived from all three germ layers, including AFP (endoderm), Tuj1 (ectodermal), and SMA (mesodermal) positive cells. Upon directed differentiation, cell clumps started beating rhythmically, and the expression of Mlc-2a and cTn I revealed that the differentiation into cardiomyocytes in these cells had been completed. Scale bar  = 200 µm (a, c).</p