Journal: Nature Communications

Article Title: An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy

doi: 10.1038/ncomms13443

Figure Lengend Snippet: ( a ) Schematic representation of self-assembled PTX/PEG 2k -Fmoc-NLG mixed micelles. ( b ) PEG 2k -Fmoc-NLG inhibited IDO enzyme activity in vitro . HeLa cells were treated with IFN-γ together with free NLG919 or PEG-NLG conjugate. Kynurenine in supernatants was measured 2 days later. Data represent means±s.e.m. * P <0.05 (versus PEG 2k -Fmoc-NLG(L), N =3), # P <0.05 (versus PEG 2k -Fmoc-NLG(S), N =3). ( c , d ) IDO1 inhibition reversed T-cell suppression mediated by IDO-expressing mouse pancreatic cancer cells (Panc02). Panc02 cells and splenocytes were mixed and treated with IL-2, anti-CD3 antibody, IFN-γ together with NLG919 or PEG-NLG conjugate for 3 days. ( c ) CD4 + and ( d ) CD8 + T-cell proliferation was examined by FACS analysis. Representative data of three independent experiments are presented as means±s.e.m. * P <0.05.

Article Snippet: Despite its reduced EC50 compared with free NLG with respect to the potency in inhibiting IDO in cultured cells, PEG-Fmoc-NLG was significantly more effective than NLG that was formulated in a similar ‘inert' nanocarrier without a NLG motif (PEG 5k -(Fmoc-Boc) 2 ) ( ).

Techniques: Activity Assay, In Vitro, Inhibition, Expressing

Journal: Nature Communications

Article Title: An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy

doi: 10.1038/ncomms13443

Figure Lengend Snippet: ( a ) PEG 2k -Fmoc-NLG(L) treatment decreased kynurenine concentrations in plasma and tumours. BALB/c mice bearing s.c. 4T1.2 tumours of ∼50 mm 3 received PBS or PEG 2k -Fmoc-NLG(L) i.v. once every 3 days for 5 times at a dose of 25 mg NLG919 per kg. Kynurenine/tryptophan ratios in plasma and tumours were determined by HPLC–MS one day following the last injection. Data are means±s.e.m. of 3 experiments. * P <0.05, ** P <0.01. ( b – g ) IDO1 inhibition by PEG 2k -Fmoc-NLG(L) increased CD4 + and CD8 + T cells, and decreased T reg cells in tumours. Tumour-bearing mice were treated as described above. ( b ) Gating of CD8 + and CD4 + T cells (marked with black boxes) as a percentage of CD45 + lymphocytes. ( c ) Gating of T reg (CD4 + FoxP3 + ) cells (marked with black boxes) as a percentage of CD4 + lymphocytes. ( d , e ) Relative number of intratumoural CD8 + ( d ) and CD4 + ( e ) T cells following different treatments. ( f , g ) Relative number of T reg cells ( f ) and CD8 + T cells/T reg ( g ) in tumour tissues. Data represent means±s.e.m. (** P <0.01, N =5). ( h ) PEG 2k -Fmoc-NLG maintained the tumour inhibitory effect. Mice bearing tumours of ∼50 mm 3 received different treatments as indicated (black arrows). * P <0.05; ** P <0.01 (versus control, N =5), # P <0.05 (versus PEG 2k -Fmoc-NLG(S), N =5). ( i ) Lymphocyte activities were required for the in vivo activity of PEG 2k -Fmoc-NLG(L) micelles. Female BALB/c-nu/nu mice bearing 4T1.2 tumour of ∼50 mm 3 were similarly treated as described above. ( j ) Enhanced in vivo antitumour activity of PEG 2k -Fmoc-NLG(L) compared with oral delivery of NLG ( # P <0.05, N =5) or NLG formulated in PEG 5k -(Fmoc-Boc) 2 micelles ( & P <0.05, N =5). * P <0.05 (versus control, N =5). Data represent means±s.e.m.

Article Snippet: Despite its reduced EC50 compared with free NLG with respect to the potency in inhibiting IDO in cultured cells, PEG-Fmoc-NLG was significantly more effective than NLG that was formulated in a similar ‘inert' nanocarrier without a NLG motif (PEG 5k -(Fmoc-Boc) 2 ) ( ).

Techniques: Clinical Proteomics, Injection, Inhibition, Control, In Vivo, Activity Assay

Journal: Nature Communications

Article Title: An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy

doi: 10.1038/ncomms13443

Figure Lengend Snippet: ( a ) Size distribution and morphology of drug-free and PTX-loaded PEG 2k -Fmoc-NLG(L) micelles (carrier: drug, 2.5:1, m/m) were examined by dynamic light scattering and TEM, respectively. Drug concentration in micelles was kept at 1 mg ml −1 . Blank micelle concentration was 20 mg ml −1 . Scale bar, 100 nm. ( b ) Measurement of CMC of PEG 2k -Fmoc-NLG(L) micelles. ( c ) PTX release kinetics of PTX/PEG 2k -Fmoc-NLG(L) examined via a dialysis method. PTX concentrations were kept at 1 mg ml −1 in PTX/PEG 2k -Fmoc-NLG(S), PTX/PEG 2k -Fmoc-NLG(L) and Taxol. PTX concentrations were analysed at 0, 1, 2, 4, 8, 24 and 48 h by HPLC. ( d ) Cytotoxicity of PEG 2k -Fmoc-NLG(L) alone, free PTX, and micellar PTX against a mouse breast cancer cell line (4T1.2) and a human prostate cancer cell line (PC3). Cells were treated for 72 h and cytotoxicity was determined by MTT assay. * P <0.05 (PTX/PEG 2k -Fmoc-NLG(L) versus PTX), N =3. ( e ) Cytotoxicity of PEG 2k -Fmoc-NLG(L) alone, free DOX, and micellar DOX against a mouse breast cancer cell line (4T1.2) and a human prostate cancer cell line (PC3). Data represent means±s.e.m.

Article Snippet: Despite its reduced EC50 compared with free NLG with respect to the potency in inhibiting IDO in cultured cells, PEG-Fmoc-NLG was significantly more effective than NLG that was formulated in a similar ‘inert' nanocarrier without a NLG motif (PEG 5k -(Fmoc-Boc) 2 ) ( ).

Techniques: Concentration Assay, MTT Assay

Journal: Nature Communications

Article Title: An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy

doi: 10.1038/ncomms13443

Figure Lengend Snippet: Biophysical characteristics of anticancer drug-loaded PEG 2K -Fmoc-NLG(L) micelles and blank micelles. *

Article Snippet: Despite its reduced EC50 compared with free NLG with respect to the potency in inhibiting IDO in cultured cells, PEG-Fmoc-NLG was significantly more effective than NLG that was formulated in a similar ‘inert' nanocarrier without a NLG motif (PEG 5k -(Fmoc-Boc) 2 ) ( ).

Techniques:

Journal: Nature Communications

Article Title: An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy

doi: 10.1038/ncomms13443

Figure Lengend Snippet: ( a , b ) The kinetics of NLG in blood ( a ) and tumour ( b ) in 4T1.2 tumour-bearing mice following i.v. administration of PEG 2k -Fmoc-NLG(L) in comparison to NLG-loaded PEG 5k -(Fmoc-Boc) 2 micelles (25 mg NLG per kg). ( c , d ) Tissue distribution of NLG in 4T1.2 tumour-bearing BALB/c mice following i.v. administration of PEG 2k -Fmoc-NLG(L) ( c ) or NLG-loaded PEG 5k -(Fmoc-Boc) 2 micelles ( d ) at a NLG dose of 25 mg kg −1 . ( e ) Blood kinetics of PTX in BALB/c mice following i.v. administration of Taxol or PTX/PEG 2k -Fmoc-NLG(L) mixed micelles at a dose of 10 mg PTX per kg. ( f ) Tissue distribution of PTX in 4T1.2 tumour-bearing BALB/c mice 24 h following i.v. administration of Taxol or PTX/PEG 2k -Fmoc-NLG(L) mixed micelles at a PTX dose of 10 mg kg −1 . * P <0.05 ( N =5). ( g , h ) Tissue distributions of PTX at various time points following i.v. administration of Taxol ( g ) or PTX/PEG 2k -Fmoc-NLG(L) mixed micelles ( h ) (10 mg PTX per kg). All data represent means±s.e.m.

Article Snippet: Despite its reduced EC50 compared with free NLG with respect to the potency in inhibiting IDO in cultured cells, PEG-Fmoc-NLG was significantly more effective than NLG that was formulated in a similar ‘inert' nanocarrier without a NLG motif (PEG 5k -(Fmoc-Boc) 2 ) ( ).

Techniques: Comparison

Journal: Nature Communications

Article Title: An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy

doi: 10.1038/ncomms13443

Figure Lengend Snippet: ( a ) In vivo antitumour activity of various PTX formulations in 4T1.2 tumour model. PTX dose was 10 mg kg −1 . Tumour sizes were plotted as relative tumour volumes. ** P <0.01 (all treatment groups versus control group), # P <0.05 (PTX/PEG 2k -Fmoc-NLG(L) versus Taxol ), & P <0.05 (PTX/PEG 2k -Fmoc-NLG(L) versus PTX/PEG 2k -Fmoc-NLG(S)). N =5. ( b ) Dose-escalation study on the antitumour activity of PTX-loaded PEG 2k -Fmoc-NLG(L) micelles. PTX dose was 5, 10 and 20 mg kg −1 , respectively. ** P <0.01 (all treatment groups versus control), # P <0.05 (10 mg, 20 mg PTX per kg versus 5 mg PTX per kg). N =5. ( c ) Antitumour activity of PTX/PEG 2k -Fmoc-NLG(L) in a 4T1.2 tumour model in comparison to a combination of oral NLG with i.v. Abraxane, PEG 2k -Fmoc-NLG(L) plus Abraxane or PEG 5k -(Fmoc-Boc) 2 micelles co-loaded with PTX and NLG. * P <0.01 (all treatment groups versus control), # P <0.05 (PTX/PEG 2k -Fmoc-NLG(L) or PEG 2k -Fmoc-NLG(L)+Abraxane versus oral NLG+Abraxane), & P <0.05 (PTX/PEG 2k -Fmoc-NLG(L) or PEG 2k -Fmoc-NLG(L)+Abraxane versus (PTX+NLG)/PEG 5k -(Fmoc-Boc) 2 ), Φ P <0.05 (PTX/PEG 2k -Fmoc-NLG(L) versus PEG 2k -Fmoc-NLG(L)+Abraxane), N =5. ( d ) Antitumour activity of PTX/PEG 2k -Fmoc-NLG(L) in a murine melanoma (B16) model. PTX dose was 10 mg kg −1 . ** P <0.01 (all treatment groups versus control), # P <0.05 (PTX/PEG 2k -Fmoc-NLG(L) versus Taxol), N=5. All data represent means±s.e.m.

Article Snippet: Despite its reduced EC50 compared with free NLG with respect to the potency in inhibiting IDO in cultured cells, PEG-Fmoc-NLG was significantly more effective than NLG that was formulated in a similar ‘inert' nanocarrier without a NLG motif (PEG 5k -(Fmoc-Boc) 2 ) ( ).

Techniques: In Vivo, Activity Assay, Control, Comparison

Journal: Nature Communications

Article Title: An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy

doi: 10.1038/ncomms13443

Figure Lengend Snippet: ( a – d ) T-cell infiltration in mouse tumours treated with Taxol, PEG 2k -Fmoc-NLG(L) or PTX/PEG 2k -Fmoc-NLG(L) at a PTX dosage of 10 mg kg −1 . The relative abundance of CD4 + , CD8 + ( a ), IFN-γ positive intratumoural CD4 + T cells ( b ), IFN-γ positive intratumoural CD8 + T cells ( c ), and granzyme B-positive CD8 + T cells ( d ) in tumour tissues were detected by flow cytometry. ( e ) Flow cytometry gating and histogram analysis of FoxP3 + T regulatory cells in mouse tumours. ( f ) Tumour-associated macrophages (TAMs) in mouse tumours. The percentages of TAM populations with specific macrophage markers (M1-type (CD11b + /F4/80 + /CD206 − ) and M2-type (CD11b + /F4/80 + /CD206 + )) in tumour tissues were detected by flow cytometry. ( g ) Flow cytometry gating and histograms analysis of CD11b + /Gr-1 + MDSC cells in mouse tumours. Double positive cells contain two populations, including Gr-1 high CD11b + granulocytic (G-MDSC) and Gr-1 int CD11b + monocytic (M-MDSC) MDSC subsets. The bars represent means±s.e.m. (* P <0.05, ** P <0.01, N =3).

Article Snippet: Despite its reduced EC50 compared with free NLG with respect to the potency in inhibiting IDO in cultured cells, PEG-Fmoc-NLG was significantly more effective than NLG that was formulated in a similar ‘inert' nanocarrier without a NLG motif (PEG 5k -(Fmoc-Boc) 2 ) ( ).

Techniques: Flow Cytometry