Porphyrin Derivative Conjugated with Gold Nanoparticles for Dual-Modality Photodynamic and Photothermal Therapies In Vitro

Gold nanoparticles (Au NPs) have been confirmed to show excellent photothermal conversion property for tumor theranostic applications. To improve the antitumor efficacy, a novel nanoplatform system composed of porphyrin derivative and Au NPs was fabricated to study the dual-modality photodynamic and photothermal therapy with laser irradiation. Modified chitosan was coated on the Au NPs surface via ligand exchange between thiol groups and Au. The chitosan-coated Au NPs (QCS-SH/Au NPs) were further conjugated with meso-tetrakis­(4-sulphonatophenyl)­porphyrin (TPPS) via electrostatic interaction to obtain the porphyrin-conjugated Au hybrid nanoparticles (TPPS/QCS-SH/Au NPs). Size, morphology, and properties of the prepared nanoparticles were confirmed by Zeta potential, nanoparticle size analyzer, transmission electron microscopy (TEM), and UV–vis spectroscopy. Moreover, both photothermal therapy (PTT) and photodynamic therapy (PDT) were investigated. Compared with alone Au NPs or TPPS, the hybrid TPPS/QCS-SH/Au NPs with lower cytotoxicity showed durable elevated temperature to around 56 °C and large amount of singlet oxygen (¹O₂) produced from TPPS. Thus, the hybrid nanoparticles showed a more significant synergistic therapy effect of hyperthermia from PTT as well as ¹O₂ from PDT, which has potential applications in the tumor therapy fields

Specificity of two linear epitopes within NS3 helicases of HCV and other flaviviruses.

<p>(A) Analysis of amino acid sequence corresponding to EP05 and EP21 regions within NS3 helicase cross HCV genotypes and other flaviviruses. Aa sequences (one-letter code) to epitopes of HCV genotype 1b are presented on the top. Positions at the beginning and end of sequences are indicated by numbers. Identities with the lead sequence are indicated by dashes. Representative sequences are retrieved from Genebank Database. Triangles labeling with EP05/2E12 or EP21/3E5 on the top indicate epitope sequences or corresponding sequences for mAb’s recognition. The aa residues GSGKS underlined in bold indicate the ATP binding site of motif I (Walker A) within NS3 helicase. n.a. indicates no corresponding sequence available from those viruses. (B and C) Reactivity of mAb 2E12 or 3E5 with mutant peptide corresponding to the defined epitope sequence in Peptide-ELISA.</p

Reactivity of epitope peptides with HCV infected plasmas.

<p>Reactivity of epitope peptides with HCV infected plasmas.</p

Overlapping peptides of HCV NS3 helicase (aa 1192–1459).

<p>Five groups of synthetic peptides derived from NS3 (1192–1459) of HCV genotype 1b (Genebank accession number AFC36922) are listed in the Table. Group A, 29 of 16mer peptides with 7mer overlapping spanning 268 amino acids of HCV NS3 helicase between aa 1192 and 1459. Group B, 6–10mer peptides are shortened from P05 or P21. Group C, peptides with an amino acid substitution from the region corresponding to P21 are derived from other genotypes of HCV variants. Group D, peptides are derived from the region of GBV-C corresponding to P05 or P21. Group E, negative control peptide (NC) is derived from <i>Brucella melitensis</i> BP26 protein. Aa, amino acid position in NS3 or whole ORF protein. Letters in the bold with underline indicate the amino acid substitution aligned with P21 or P05.</p

Classification of mAbs reacting with HCV NS3 helicase.

<p>Aa, amino acid position; L, linear; SC, semi-conformational; C, conformational; UN, un-classified; NT, not tested; the native NS3 indicates NS3 produced from HCV JFH-1 infected cells;</p>*<p>indicates mAb 3E5 reactive to both T1b-rNS3 with 347I and FL4b-rNS3 with 347V in ELISA.</p

Unwinding activity of HCV NS3 helicase inhibited by mAb 2E12.

<p>(A) A representative of DNA oligonucleotides labeled with Fluorescein amidite (FAM) from unwound double-strand DNA substrates at various concentrations of NS3 helicase of HCV (FL4b-rNS3) in unwinding reactions. (B) The slope of the initial velocity curve calculated from 4 representative tests with mean±SD. (C) A representative of unwinding reactions with 75 nM NS3 helicase and 1 µg/ml mAb 2E12 or an unrelated control mAb to <i>B. melitensis</i>. (D) Percentages for unwinding activity measured as velocity from 4 representative tests of unwinding reactions with NS3 helicase and mAbs.</p

Reactivity of mAbs to peptides and proteins of NS3 helicase.

<p>MAbs reacted with 16mer peptides in Peptide-ELISA (A), denatured T1b-rNS3 in Western Blot (B), denatured FL1b-rNS3 expressing 293T cells (C. I) and denatured native NS3 of HCV JFH-1 (2a) infected Huh7.5.1 cells (D. I) in Western Blot, and non-denatured native NS3 of HCV JFH-1 infected Huh7.5.1 cells in IFS (E). C. II and D. II, un-transfected 293T or un-infected Huh7.5.1 cell controls, respectively; NC (negative control), an un-related mAb to BP26 protein of <i>B. melitensis</i>; (+), mAb C7–50 to HCV core as positive control; (−), un-infected Huh7.5.1 cells as negative control.</p

Characterizing the dynamics of BCR repertoire from repeated influenza vaccination

Yearly epidemics of seasonal influenza cause an enormous disease burden around the globe. An understanding of the rules behind the immune response with repeated vaccination still presents a significant challenge, which would be helpful for optimizing the vaccination strategy. In this study, 34 healthy volunteers with 16 vaccinated were recruited, and the dynamics of the BCR repertoire for consecutive vaccinations in two seasons were tracked. In terms of diversity, length, network, V and J gene segments usage, somatic hypermutation (SHM) rate and isotype, it was found that the overall changes were stronger in the acute phase of the first vaccination than the second vaccination. However, the V gene segments of IGHV4-39, IGHV3-9, IGHV3-7 and IGHV1-69 were amplified in the acute phase of the first vaccination, with IGHV3-7 dominant. On the other hand, for the second vaccination, the changes were dominated by IGHV1-69, with potential for coding broad neutralizing antibody. Additional analysis indicates that the application of V gene segment for IGHV3-7 in the acute phase of the first vaccination was due to the elevated usage of isotypes IgM and IgG3. While for IGHV1-69 in the second vaccination, it was contributed by isotypes IgG1 and IgG2. Finally, 41 public BCR clusters were identified in the vaccine group, with both IGHV3-7 and IGHV1-69 were involved and representative complementarity determining region 3 (CDR3) motifs were characterized. This study provides insights into the immune response dynamics following repeated influenza vaccination in humans and can inform universal vaccine design and vaccine strategies in the future.</p