Functionalization of immunostimulating complexes (ISCOMs) with lipid vinyl sulfones and their application in immunological techniques and therapy

Figures & data

Figure 1 Synthesis of vinyl-sulfone reagents for the functionalization of nanocapsules. (i) HS(CH₂)₂OH, K₂CO₃, DMSO–THF (1:1), rt, 16 hours. (ii) H₂O₂, AcOH, rt, 16 hours. (iii) methanesulfonyl chloride, Et₃N, CH₂Cl₂ anh, rt, 24 hours. (iv) (a) Cl₂SO, rt, 1 hour; (b) cystamine, Et₃N, CH₂C_l2 anh, rt, 15 minutes. (v) (a) Zn, AcOH, 50°C, 40 minutes; (b) DVS, Et₃N, THF-iPrOH (2:1), rt, 90 minutes. (vi) DVS, tBuOK, THF, rt, 1 hour.

Abbreviations: AcOH, acetic acid; DMSO, dimethyl sulfoxide; DVS, divinyl sulfone; rt, room temperature; THF, tetrahydrofuran.

Figure 2 Transmission electron microscope images. (A) ISCOM-matrix. (B) ISCOM-phy-7-A-IgG-specific. (C) ISCOM-4-A-IgG-specific containing actinomycin D. (D) ISCOM-9-A-IgG specific IgGs.

Note: Bar = 150 nm.

Abbreviations: IgG, immunoglobulin; ISCOM, immunostimulating complex.

Figure 3 Laser confocal micrographs of epimastigotes of Trypanosoma cruzi incubated with different phycocyanin nanocapsules. (A) ISCOM-phy; (B) ISCOM-phy-4-A; (C) ISCOM-phy-4-A-IgGsp; (D) ISCOM-phy-7-A; (E) ISCOM-phy-7-A-IgGsp.

Notes: (1) Laser confocal and (2) phase contrast microscope images.

Abbreviations: IgG, immunoglobulin; IgGsp, IgG specific; ISCOM, immunostimulating complex; phy, phycocianin.

Figure 4 Evaluation of bound IgGs.

Notes: Determination with ELISA of the immunoglobulins bound to the nanoparticles via a secondary anti-mouse antibody labeled with peroxidase. The results are the mean of three experiments ± standard error of the mean. **P < 0.01 Tukey–Kramer multiple comparisons test; ***P < 0.001. 4, ISCOM with compound 4 linked to mouse Prot A-IgG; 7, ISCOM with compound 7 linked to Prot A bound to mouse IgG; 9, ISCOM formed with compound 9 (1:1) linked to Prot A bound to mouse IgG; ISC matrix, ISCOM-matrix; Isc Prt A H IgG, ISCOM with compound 4 linked to Prot A and human IgG; IgG spec, mouse IgG obtained after immunization with Trypanosoma cruzi.

Abbreviations: IgG, immunoglobulin; ISCOM, immunostimulating complex; OD, optical density; Prot A, protein A.

Table 1 Relative fluorescence of the different immunostimulating complex (ISCOM) preparations

Sample	Relative fluorescence (mean ± SD)
ISCOM-phy	0.034 ± 0.01
ISCOM-phy7-A	0.058 ± 0.02
ISCOM-phy4-A	0.05 ± 0.01
ISCOM-phy7-A-IgGsp	16.477 ± 0.45***
ISCOM-phy4-A-IgGsp	14.03 ± 0.12***
ISCOM-9-phy-Aa	0.02 ± 0.01
ISCOM-9-phy-A-IgGspa	19.501 ± 0.31***
ISCOM-9-phy-Ab	0.03 ± 0.01
ISCOM-9-phy-A-IgGspb	12.09 ± 0.12***
ISCOM-phy7-A-IgG non-sp	0.5 ± 0.16
ISCOM-phy4-A-IgG non-sp	0.03 ± 0.01
ISCOM-9-phy-A-IgG non-spa	0.058 ± 0.02

Note:

*** P < 0.001 by the Tukey–Kramer multiple comparisons test, between the samples without specific G immunoglobulin (IgG).

Abbreviations: IgG, immunoglobulin; ISCOM-phy, ISCOM labelled with phycocyanin; ISCOM-phy 7-A, protein A bound to ISCOM-phy with compound 7; ISCOM-phy 4-A, protein A bound to ISCOM-phy with compound 4; ISCOM-phy 7-A-IgGsp, ISCOM-phy, compound 7 with protein A and Trypanosoma cruzi-specific mouse IgG; ISCOM-phy 4-A-IgGsp, ISCOM-phy, compound 4 with protein A and T. cruzi-specific mouse IgG; ISCOM-9-phy-Aa, ISCOM-phy prepared with modified cholesterol (compound 9 at a ratio of 1:1) and protein A; ISCOM-9-phy-A-IgGspa, ISCOM-phy prepared with modified cholesterol (compound 9 at a ratio of 1:1) and protein A and T. cruzi-specific mouse IgG; ISCOM-9-phy-Ab, ISCOM-phy prepared with modified cholesterol (compound 9 at a ratio of 2:3) with protein A; ISCOM-9-phy-A-IgGspb, ISCOM-phy prepared with modified cholesterol (compound 9 at a ratio of 2:3) with protein A and T. cruzi-specific mouse IgG; ISCOM-phy 7-A-IgG non-sp ISCOM-phy prepared with compound 7, protein A and nonspecific human IgG; ISCOM-phy 4-A-IgG non-sp, ISCOM-phy prepared with compound 4, protein A and nonspecific human IgG; ISCOM-9-phy-A-IgG non-sp, ISCOM-phy prepared with modified cholesterol (compound 9 at a ratio of 2:3), protein A and nonspecific human IgG.

Figure 5 Laser-confocal micrograph of epimastigotes of Trypanosoma cruzi incubated with nanocapsules formed with compound 9. (A) ISCOM-9-phy-A (1:1). (B) ISCOM-9- phy-A-IgGsp (1:1). (C) ISCOM-9-phy-A (2:3). (D) ISCOM-9-phy-A-IgGsp (2:3).

Notes: Immunostimulating complex (ISCOM)-9-phycocianin (phy)-A (1:1), nanocapsules formed with compound 9–cholesterol (1:1) with phy linked to protein A (Prot A) without specific G immunoglobulin (IgG); ISCOM-9-phy-A-IgGsp (1:1), nanocapsules formed with compound 9–cholesterol (1:1) with phycocianin linked to specific Prot A IgG; ISCOM-9-phy-A(2:3), nanocapsules formed with compound 9–cholesterol (2:3) with phycocianin linked to Prot A without specific IgG; ISCOM-9-phy-A-IgGsp (2:3), nanocapsules formed with compound 9–cholesterol (2:3) with phycocianin linked to Prot A specific IgG; 1, Laser confocal; 2, phase contrast microscope images.

Figure 6 Cytotoxicity + against epimastigote forms of Trypanosoma cruzi measured after 24 hours treatment with actinomycin D contained in functionalized nanocapsules bound to IgGs. (A) Epimastigotes of T. cruzi treated with 50 ng/mL of actinomycin D. (B) Epimastigotes of T. cruzi treated with ISCOM-9-actinomycin D-A-IgGsp containing 0.15 × 10⁻² ng/mL of actinomycin D. (C) Epimastigotes of T. cruzi treated with ISCOM-9-actinomicyn D-A-IgGsp containing 2.88 × 10⁻² ng/mL of actinomycin D. (D) Epimastigotes of T. cruzi treated with ISCOM-9- actinomycin D-A-IgGsp containing 5.76 × 10⁻² ng/mL of actinomycin D. (E) Control culture of untreated epimastigote forms of T. cruzi. (F) Control culture of epimastigote forms of T. cruzi treated with functionalized nanocapsules without actinomycin D ISCOM-9-matrix A-IgG specific against the parasite.

Notes: The results are the mean of three experiments ± standard error of the mean. ns = P > 0.05; ***P < 0.001 Tukey–Kramer multiple comparisons test.

Abbreviations: IgG, immunoglobulin; ISCOM, immunostimulating complex; ns, not significant.

Figure 7 Schematic representation of the ISCOM with compounds 4, 7, or 9 linked to protein A.

Abbreviations: ISCOM, immunostimulating complex; Prot A, protein A.

Figure 8 Graphical abstract of the ISCOM with the compounds linked to protein A–IgG.

Abbreviations: IgG, immunoglobulin; IgGsp, IgG specific; ISCOM, immunostimulating complex; Prot A, protein A; T. cruzi, Trypanosoma cruzi.

Figure S1 ¹H NMR spectra for compound 4.

Figure S2 ¹³C and DEPT NMR spectra for compound 4.

Figure S3 ¹H NMR spectra for compound 7.

Figure S4 ¹³C and DEPT NMR spectra for compound 7.

Figure S5 ¹H NMR spectra for compound 9.

Figure S6 ¹³C and DEPT NMR spectra for compound 9.