Co-delivery of carbonic anhydrase IX inhibitor and doxorubicin as a promising approach to address hypoxia-induced chemoresistance

Figures & data

Scheme 1. Representation of lipid coated mesoporous silica nanoparticle for co-delivery of carbonic anhydrase IX inhibitor and doxorubicin in hypoxia to overcome chemoresistance.

Figure 1. Image representing (a) FTIR spectrums of CTAB, CTAB-MSNPs and MSNPs, (b) MSNPs Nitrogen Adsorption-desorption isotherms before and after washing (c) BJH graph showing the pore size.

Table 1. Surface area, pore size and volume of MSNPs before and after surfactant removal.

Samples	Surface Area (m^2/g)	Vm (cm^3(STP) /g)	Pore diameter d-peak (nm)	Total Pore Vol (cm^3/g)
CTAB-MSNPs	42.75	9.80	2.43	0.34
MSNPs	1134.43	260.61	2.43	1.07

Figure 2. TEM images of (a-b) MSNPs showing the porous structure and (b) (i & ii) are representing FFT and IFFT patterns respectively.

Figure 3. AFM images showing (a & d) amplitude trace, (b & e) height trace, (c & f) respective identified line graph and G as a representative image of surface measurement of lipid coating to MSNPs.

Figure 4. Doxorubicin release profile from Dox-MSNPs and Lip_c-Dox-MSNPs.

Table 2. Physicochemical parameter changes of formulations after incubating in PBS, medium with 10% and 60% FCS at 37 °C and 100 rpm for 4 h and 24 h. The hydrodynamic size (intensity distribution), PDI and zeta potential are presented as mean ± SD (n = 3).

Medium	Time (h)	Lipc			Lipc-Dox-MSNPs
		Avg. Size (nm) ± SD	PDI ± SD	Zeta. Pot (mV) ± SD	Avg. Size (nm) ± SD	PDI ± SD	Zeta. Pot (mV) ± SD
PBS	0	109.2 ± 9.69	0.21 ± 0.01	23.50 ± 7.51	126.39 ± 8.73	0.28 ± .02	14.26 ± 3.32
	4	110.91 ± 4.65	0.23 ± 0.01	20.44 ± 4.26	134.68 ± 12.83	0.26 ± 0.02	3.75 ± 0.61
	24	133.2 ± 2.5	0.26 ± 0.04	5.0 ± 0.57	139.23 ± 2.25	0.30 ± 0.01	4.8 ± 1.42
Medium + 10% FCS	0	109.2 ± 9.69	0.21 ± 0.01	23.50 ± 7.51	126.39 ± 8.73	0.28 ± .02	14.26 ± 3.32
	4	116.95 ± 3.23	0.235 ± 0.01	4.26 ± 0.70	147.36 ± 9.72	0.229 ± 0.06	4.33 ± 0.80
	24	113.97 ± 7.78	0.23 ± 0.01	3.575 ± 1.38	149.23 ± 10.0	0.28 ± 0.016	2.44 ± 1.51
Medium + 60% FCS	0	109.2 ± 9.69	0.21 ± 0.01	23.50 ± 7.51	126.39 ± 8.73	0.28 ± .02	14.26 ± 3.32
	4	185.3 ± 65.74	0.33 ± 0.06	2.58 ± 1.55	121.13 ± 8.7	0.25 ± 0.02	1.98 ± 1.48
	24	220.17 ± 23.35	0.46 ± 0.18	−3.12 ± 7.56	147.93 ± 31.93	0.33 ± .03	0.11 ± 1.44

Figure 5. Showing (a) Western blots of CA-IX and HIF-1α expression under normoxia and hypoxia, (b) CA-IX expression after treatment, (c and d) Extracellular acidification in normoxia and hypoxia before and after traetment with CAI, Lip_c and Lip_c-Dox-MSNPs after 4 h and 24 h incubation respectively. Values are represented as mean ± SD (n = 3) and statistical significance p values (p < 0.05) are presented as ***p < 0.001, **p < 0.01, *p < 0.1 and ^nsp = non-significant.

Figure 6. In vitro cytotoxicity evaluation of (a) Dox (4 h & 24 h incubation) with CAI and Lip_c pretreated cells in normoxia and hypoxia and (b) Lip_c-Dox-MSNPs, Lip-Dox-MSNPs and Dox after 4 h & 24 h incubation in normoxia and hypoxia. Values are represented as mean ± SD (n = 3) and statistical significance p values (p < 0.05) are presented as ***p < 0.001, **p < 0.01, *p < 0.1 and ^nsp = non-significant.

Figure 7. In vitro cellular uptake of doxorubicin where (a) is the representing qualitative uptake of Dox with different formulations in normoxia and hypoxia observed witgh CLSM and (b) Quantitaive evaluation of mean fluorescenc intensity (MFI) of Dox uptake with different formulation measured with FACS. Values are represented as mean ± SD (n = 3) and statistical significance p values (p < 0.05) are presented as ****p < 0.0001.

Figure 8. Hemocmpatibility assay showing (a) coagulation time and (b) hemolysis effects of different formulations and free Dox. Tritox-X100 and blood plasma were considered as negative and positive control. All the values are represented as mean of triplicate along with standard deviation. Values are represented as mean ± SD (n = 3) and statistical significance p values (p < 0.05) are presented as ****p < 0.0001, ***p < 0.001, **p < 0.01.

Data availability statement

The data that support the findings of this study are available from the corresponding author, U.B., upon reasonable request.