Targeted delivery of doxorubicin-utilizing chitosan nanoparticles surface-functionalized with anti-Her2 trastuzumab

Figures & data

Figure 1 Schematic illustration of the procedures for the synthesis of chitosan-doxorubicin conjugate.

Abbreviations: EDC, 1-ethyl-3-(3-dimethyl amino-propyl) carbodiimide hydrochloride; NHS, N-hydroxysuccinimide.

Table 1 Drug content and conjugation efficiency of different chitosan-doxorubicin conjugates

Conjugation efficiency	Drug content	DOX used/CS (w/w)	Conjugate
33%	6.7%	20%	CS-DOX-1
41%	4.1%	10%	CS-DOX-2
54.6%	2.37%	5%	CS-DOX-3
62%	1.55%	2.5%	CS-DOX-4
69%	0.69%	1%	CS-DOX-5

Abbreviations: CS, chitosan; DOX, doxorubicin.

Figure 2 (A) Fourier transform infrared spectrum of doxorubicin and succinyl doxorubicin. (B) ¹H nuclear magnetic resonance spectrum of doxorubicin and succinyl doxorubicin. (C) ¹H nuclear magnetic resonance spectrum of chitosan and chitosan-doxorubicin conjugate.

Figure 3 Differential scanning calorimetry thermograms of chitosan, doxorubicin and chitosan-doxorubicin conjugate (CS-DOX-2).

Figure 4 Gel permeation chromatogram of (A) chitosan and (B) chitosan-doxorubicin conjugates.

Table 2 Size, zeta potential, and polydispersity index of nanoaggregates prepared from different chitosan-doxorubicin conjugates

PDI	Zeta potential (mV)	Size (nm)	Drug content	Conjugate
0.108 ± 0.054	16.5 ± 3	228 ± 21	4.1%	CS-DOX-2
0.173 ± 0.041	17.5 ± 4	346 ± 37	2.37%	CS-DOX-3
0.083 ± 0.047	14.3 ± 1	493 ± 26	1.55%	CS-DOX-4
0.253 ± 0.023	18.1 ± 4	671 ± 18	0.69%	CS-DOX-5

Abbreviations: CS-DOX, chitosan-doxorubicin conjugate; PDI, polydispersity index.

Figure 5 Effect of chitosan-doxorubicin conjugate concentration on hydrodynamic diameter of nanoaggregates.

Abbreviation: CS-DOX, chitosan-doxorubicin conjugate.

Figure 6 (A) Scanning electron micrographs of chitosan-doxorubicin conjugate (CS-DOX-2) nanoparticles and (B) transmission electron micrographs of CS-DOX- 2 nanoparticles.

Figure 7 Isothermal titration calorimetry thermograms for interactions of protein A with (A) free antibody and (B) antibody attached to nanoparticles.

Table 3 Interaction thermodynamic parameters of protein A with free and attached antibody

Nanoparticle-conjugated monoclonal antibody		Free monoclonal antibody	Parameter
Second binding site	First binding site
1.07 ± 0.261	1.65 ± 0.099	2.78 ± 0.469	an
203.6	348.1	5.71	bK (×10^−4 M^−1)
+68.1	−93.9	−104.8	cΔH°(kcal/mol)
−8.664	−10.162	−6.96	dΔG°(kcal/mol)
+0.318	−0.281	−0.330	eΔS°(kcal/mol/°K)

Notes:

a binding stoichiometry;

b affinity constant;

c molar enthalpy change for the binding;

d molar free energy change;

e molar entropy change.

Figure 8 Effect of doxorubicin, chitosan-doxorubicin conjugated nanoparticles, and trastuzumab-decorated chitosan-doxorubicin conjugated nanoparticles at different concentrations on viability of SKOV-3 cell line.

Abbreviations: CS, chitosan; DOX, doxorubicin; NP, nanoparticles.

Figure 9 Fluorescence microscopy of cellular uptake of (A) trastuzumab-decorated chitosan-doxorubicin conjugated nanoparticles by SKOV-3 cell line, (B) trastuzumab-decorated chitosan-doxorubicin conjugated nanoparticles by MCF-7 cell line, (C) chitosan-doxorubicin conjugated nanoparticles by SKOV-3 cell line, and (D) chitosan-doxorubicin conjugated nanoparticles by MCF-7 cell line. (E) SKOV-3 cells and (F) MCF-7 cells in cell culture medium served as negative controls.

Figure 10 Schematic illustration of self-assembly of chitosan-doxorubicin conjugates into nanoparticles.

Figure 11 Interaction of protein A with antibodies attached to the nanoparticle surface.

Abbreviation: CS-DOX, chitosan-doxorubicin conjugate.