T cells Proliferation by purified DC-tumor hybrids or other components of the fusion products.

<p>Non-adherent PBMCs from the same patient (from patient1) were purified using nylon wool to remove antigen-presenting cells and B cells. They were incubated with purified hybrid cells, adherent tumor cells, the non-adherent cell fraction, the adherent cell fraction, purified hybrid cells supplemented with non-adherent cell fraction, total fusion products, DCs mixed with tumor cells, or DCs at a ratio of 10∶1 in the presence of 20 units/ml human IL-2. T cells cultured in the presence of 20 units/ml human IL-2 were used as a control. T cell proliferation was determined using the standard [³H] thymidine uptake assay. <i>Columns</i>, mean values of triplicate samples; <i>bars</i>, SD. *<i>P</i><0.05 for T cell proliferation stimulated by purified hybrids, the adherent cell fraction, the non-adherent cell fraction, total fusion products or purified hybrid cells supplemented with the non-adherent cell fraction compared with adherent tumor cells, DCs mixed with tumor cells, DCs, or IL-2 alone. **<i>P</i><0.05 for T cell proliferation stimulated by the total fusion products, purified hybrid cells supplemented with the non-adherent cell fraction, purified hybrid cells or the adherent cell fraction compared with the non-adherent cell fraction. ***<i>P</i><0.05 for T cell proliferation stimulated by the total fusion products or purified hybrid cells supplemented with the non-adherent cell fraction compared to purified hybrid cells or the adherent cell fraction. There was no difference between the total fusion products and purified hybrid cells supplemented with the non-adherent cell fraction or between purified hybrids and the adherent cell fraction (<i>P</i>>0.05).</p

FACS analysis of apoptotic/necrosis tumor cells phagocytosed by DCs.

<p>DCs and auto breast tumor cells (from patient 1) were stained red and green by PKH26 and PKH67 and double positive cells were analyzed using FACS and confocal microscopy. A, FACS analysis of the non-adherent cells fraction from the total fusion products. DC phagocytosis of apoptotic tumor cells was calculated as the percentage of double-positive cells, and was approximately 42%. B, FACS analysis of DCs mixed with tumor cells. No significant phagocytosis was observed.</p

Ultrathin Polypyrrole Nanosheets via Space-Confined Synthesis for Efficient Photothermal Therapy in the Second Near-Infrared Window

Extensive efforts have been devoted to synthesizing photothermal agents (PTAs) that are active in the first near-infrared (NIR) region (650–950 nm). However, PTAs for photothermal therapy in the second NIR window (1000–1350 nm) are still rare. Here, it is shown that two-dimensional ultrathin polypyrrole (PPy) nanosheets prepared via a novel space-confined synthesis method could exhibit unique broadband absorption with a large extinction coefficient of 27.8 L g^–1 cm^–1 at 1064 nm and can be used as an efficient PTA in the second NIR window. This unique optical property is attributed to the formation of bipolaron bands in highly doped PPy nanosheets. The measured prominent photothermal conversion efficiency could achieve 64.6%, surpassing previous PTAs that are active in the second NIR window. Both in vitro and in vivo studies reveal that these ultrathin PPy nanosheets possess good biocompatibility and notable tumor ablation ability in the second NIR window. Our study highlights the potential of ultrathin two-dimensional polymers with unique optical properties in biomedical applications

CTL assays induced by purified DC-tumor hybrids or other components of the fusion products.

<p>The cytotoxicity assays were performed using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. (A, C, E, G) Non-adherent PBMCs stimulated with total fusion products (▪), hybrid cells supplemented with the non-adherent cell fraction (◊), purified hybrid cells (○), the adherent cell fraction (▴), the non-adherent cell fraction (△), adherent tumor cells (♦), or DCs mixed with tumor cells (□) for 7 days in the presence of 20 units/mL human IL-2 were used as the effector T cells. Then breast tumor cells were co-cultured with the effector T cells for 4 h at ratios of 1∶12.5, 1∶25, and 1∶50, respectively. <i>Points</i>, mean values of triplicate samples; <i>bars</i>, SD. The results showed that T lymphocytes activated by purified hybrids, the adherent cell fraction, the non-adherent cell fraction, total fusion products, purified hybrid cells supplemented with the non-adherent cell fraction lysed auto breast tumor cells much more effectively than T cells activated by adherent tumor cells or DC mixed with tumor cells (<i>P</i><0.05). Lysis induced by total fusion products or purified hybrid cells supplemented with the non-adherent cell fraction was the most effective. Purified hybrid cells or the adherent cell fraction induced more effective lysis than the non-adherent cell fraction (<i>P</i><0.05). There was no difference between total fusion products and purified hybrid cells supplemented with the non-adherent cell fraction or between purified hybrids and the adherent cell fraction (<i>P</i>>0.05). (B, D, F) Natural killer-sensitive K562 cells and monocytes were used as control targets in a parallel CTL assay, and the ratio for effector and target cells was 50∶1. <i>Columns</i>, mean values of triplicate samples; <i>bars</i>, SD. No lysis against K562 or monocytes was induced. H, T cells were stimulated by purified hybrid cells supplemented with the non-adherent cell fraction and then normal breast tumor cells lines MCF7, SKBR3 and BT20 were included as targets and MHC Class I molecule blocking test was done. Effector T can not only lyse the auto breast tumor cells (HLA A2+/A11−, HER2+), but also lyse the HLA-A2 matched MCF7 (HLA A2+/A11−, HER2+) to a less extent ( P<0.05) which can be blocked by preincubation with anti-MHC I antibody. (A B from patient1, C D from patient2, E F from patient3, G H from patient4).</p

IFN-gamma production stimulated with purified DC/tumor hybrids or other components of the fusion products.

<p>Non-adherent PBMCs, purified using nylon wool, were co-cultured with purified hybrid cells, adherent tumor cells, the non-adherent cell fraction, the adherent cell fraction, purified hybrid cells supplemented with the non-adherent cell fraction, total fusion products, or DCs mixed with tumor cells at a ratio of 10∶1 in complete RPMI 1640 for 7 days and then harvested as effector cells using nylon wool separation. Patient-derived tumor cells were used as stimulator cells. IFN-gamma production was determined using a human IFN-gamma ELISPOT kit. A medium-only control and tumor cells without effector cells were included as negative controls. <i>Columns</i>, mean values of triplicate samples; <i>bars</i>, SD. *<i>P</i><0.05 for the number of IFN-gamma positive T cells induced by purified hybrids, the adherent cell fraction, the non-adherent cell fraction, total fusion products or purified hybrid cells supplemented with the non-adherent cell fraction compared to DCs mixed with tumor cells. **<i>P</i><0.05 for the number of IFN-gamma positive T cells induced by total fusion products, purified hybrid cells supplemented with the non-adherent cell fraction, purified hybrid cells or the adherent cell fraction compared with non-adherent cell fraction. ***<i>P</i><0.05 for the number of IFN-gamma-positive T cells induced by total fusion products or purified hybrid cells supplemented with the non-adherent cell fraction compared with purified hybrid cells or the adherent cell fraction. There was no difference between total fusion products and purified hybrid cells supplemented with the non-adherent cell fraction or between purified hybrids and the adherent cell fraction (<i>P</i>>0.05). (A from patient1, B from patient2, C from patient3).</p

DC maturation analysis after phagocytosis of apoptotic/necrotic tumor cells.

<p>The non-adherent cell fraction from DC/tumor fusion products or from the DC tumor mixture (from patient1) was stained with fluorescence labeled antibody against MHC class II, CD80, CD86 and CD83 and FACS analysis was performed. A, Non-adherent cells (containing mainly unfused DCs) from the total fusion products demonstrated significant MHC class II (increased fluorescence intensity), CD80, CD86 and CD83 up-regulation. B, Up-regulation of these molecules in DCs cocultured with tumor cells was minimal. In each histogram the percentage of positive cells is indicated.</p

Antitumor immune responses of DCs with phagocytic tumor cells in non-adherent cell fraction.

<p>DCs and DCs with phagocytic tumor cells in the non-adherent cells fraction were sorted and antitumor immune response were compared through Ellispot and CTL assay (from patient4). Compared with DCs, DCs with phagocytic tumor cells can stimulate more IFN-Gamma secretion (A, P<0.05) and much more powerful CTL responses (B, P<0.05) and the immune responses were not as strong as fusion hybrids (P<0.05).</p

FACS analysis of electro-fusion between auto DCs and tumor cells.

<p>Prior to fusion, DCs and tumor cells were stained red and green (from patient 4), respectively, using the PKH26-GL and PKH67-GL kits. Tumor cells were then irradiated with 5000 cGy, and standard electro-fusion was performed on the DCs and tumor cells at a ratio of 2∶1. The fusion mixture was incubated overnight in a CO₂ tissue culture incubator. On the second day, cells were collected and subjected to FACS analysis. The red DCs and green tumor cells were clearly distinct(A, B). The amount of double-positive hybrid cells in adherent cell population was 47% (C), and 97% in the purified hybrids cell population (D).</p

Dipole Orientation Matters: Longer-Circulating Choline Phosphate than Phosphocholine Liposomes for Enhanced Tumor Targeting

Zwitterionic phosphocholine (PC) liposomes are widely used in drug delivery because of their high biocompatibility and long blood circulation time. We herein report that by flipping the direction of the PC dipole, the resulting choline phosphate (CPe) liposomes have an even longer circulation time, as confirmed at both cellular and animal-model levels. Even when 33% cholesterol was included in the lipid formulation with a poly­(ethylene glycol) layer, the CPe liposome still had a longer blood circulation time. Isothermal titration calorimetry indicates a lack of protein adsorption or PC membrane attachment for the CPe liposomes. This is different from the previously reported adhesion of CP polymers to PC lipid membranes, which may be attributed to the different ways of displaying the CP headgroup. With a longer blood circulation time, the CPe liposomes accumulated in tumors more easily than PC liposomes, which is likely due to the enhanced permeation and retention effect and tumor cell uptake. This study provides key insights into zwitterionic biointerfaces for biomedical, analytical, and materials applications

Revealing the Cytotoxicity of Residues of Phosphazene Catalysts Used for the Synthesis of Poly(ethylene oxide)

We herein report a case study on the toxicity of residual catalyst in metal-free polymer. Eight-arm star-like poly­(ethylene oxide)­s were successfully synthesized via phosphazene-catalyzed ring-opening polymerization of ethylene oxide using sucrose as an octahydroxy initiator. The products were subjected to MTT assay using human cancer cell lines (MDA-MB-231 and A2780). Comparison between the crude and purified products clearly revealed that the residual phosphazenium salts were considerably cytotoxic, regardless of the anionic species, and that the cytotoxicity of more bulky <i>t</i>-BuP₄ salt was higher than that of <i>t</i>-BuP₂ salt. Such results have therefore put forward the necessity for removal of the catalyst residues from PEO-based polymers synthesized through phosphazene catalysis for biorelated applications and for the development of less or nontoxic organocatalysts for such polymers