Targeting pituitary adenomas with folate-conjugated multiple drug decorated liposomal formulations for improved antiproliferative anticancer efficacy

Figures & data

Figure 1. FTIR spectra of free temozolomide, free cabergoline, FOL-DUAL-ILs, FOL-DUAL-DLs, UN-DUAL-DLs and FOLATE-DSPE conjugate.

Table 1. Size distribution, polydispersity index (PDI) and Zeta potential of the liposomal formulations.

Sl. No.		Size of nanoparticles (nm)	Zeta potential (mV)	Polydispersity index (PDI)
1.	FOL-DUAL-ILs	262.3 ± 2.2	27.1 ± 0.2	0.082 ± 0.02
2.	FOL-DUAL-DLs	422.9 ± 1.2	27.6 ± 0.1	0.085 ± 0.01
3.	UN-DUAL-DLs	411.2 ± 1.2	21.5 ± 0.3	0.075 ± 0.02

Table 2. EE (%) for temozolomide and cabergoline in the liposomal formulations.

Sl. No.		EE (%) for temozolomide	EE (%) for cabergoline
1.	FOL-DUAL-ILs	56.2 ± 2.3	51.3 ± 1.7
2.	FOL-DUAL-DLs	79.7 ± 2.9	75.3 ± 2.7
3.	UN-DUAL-DLs	72.7 ± 4.1	66.3 ± 1.5

Figure 2. Phase contrast microscopy images: 2A FOL-DUAL-ILs; 2B FOL-DUAL-DLs; 2C UN-DUAL-DLs.

Figure 3. CRYO-TEM microscopy images: 3 A FOL-DUAL-ILs; 3B FOL-DUAL-DLs; 3C UN-DUAL-DLs.

Table 3. Size, zeta potential and PDI of FOL-DUAL-ILs, FOL-DUAL-DLs and UN-DUAL-DLs after 10, 20 & 30 days during stability studies.

	FOL-DUAL-ILs			FOL-DUAL-DLs			UN-DUAL-DLs
	Size (nm)	Zeta potential (mV)	PDI	Size (nm)	Zeta potential (mV)	PDI	Size (nm)	Zeta potential (mV)	PDI
10 Days	161.2 ± 1.2	−26.3 ± 1.4	0.082 ± 0.07	420.13 ± 1.6	−26.6 ± 0.3	0.084 ± 0.03	410.13 ± 0.3	−21.2 ± 0.1	0.074 ± 0.04
20 days	159.4 ± 1.5	−25.3 ± 1.1	0.082 ± 0.06	419.33 ± 1.8	−26.1 ± 0.5	0.083 ± 0.01	409.41 ± 1.5	−20.6 ± 0.1	0.0.74 ± 0.01
30 Days	158.6 ± 1.1	−24.5 ± 0.1	0.81 ± 0.03	417.13 ± 0.2	−24.3 ± 0.5	0.082 ± 0.03	409.10 ± 1.4	−20.3 ± 0.2	0.072 ± 0.02

Figure 4. In vitro release studies of temozolomide and cabergoline respectively 4 A & 4B pH 7.4 (normal physiological milieu) 4 C & 4 D pH 5.0 (tumor milieu).

Table 4. Haematological parameters observed in rats post intravenous inject of the free drugs, mixture and liposomal formulations.

Sl. No.	Formulation	WBC (x 10^3/µL)	RBC (x 10^3/µL)	Haemoglobin (g/dL)	Platelet Count (x 10^3/µL)
1.	Free Temozolomide	2.8 ± 0.4	6.2 ± 0.4	10.6 ± 1.2	867.5 ± 32.4
2.	Free Cabergoline	3.4 ± 0.6	8.7 ± 0.6	11.2 ± 1.3	880.2 ± 31.6
3.	Temozolomide & cabergoline mixture	2.4 ± 0.2	7.1 ± 0.4	11.5 ± 1.1	792.5 ± 34.3
4.	FOL-DUAL-ILs	7.5 ± 1.3	7.9 ± 1.4	14.1 ± 0.6	915.3 ± 31.9
5.	FOL-DUAL-DLs	7.8 ± 0.2	9.1 ± 0.8	16.1 ± 1.0	995.4 ± 32.7
6.	UN-DUAL-DLs	7.4 ± 0.6	7.5 ± 1.6	14.5 ± 1.6	915.3 ± 31.9
7.	PBS (Control)	7.4 ± 1.2	9.0 ± 0.5	15.8 ± 1.8	1012.6 ± 32.6

Figure 5. Qualitative uptakes of the free drugs and liposomal formulations by primary cell cultures 5 A free temozolomide; 5B free cabergoline; 5 C cabergoline and temozolomide mixture; 5 D FOL-DUAL-ILs; 5E FOL-DUAL-DLs; 5 F UN-DUAL-DLs.

Figure 6. Quantitative uptake was elaborated by FACS assay of free drugs and liposomal formulations by primary cell cultures.

Figure 7. % Cellular viability on being treated with FOL-DUAL-DLs, FOL-DUAL-ILs, UN-DUAL-DLs, PBS (control), free temozolomide, free cabergoline and temozolomide-cabergoline mixture.

Figure 8. Cellular viability on being treated with the following formulations 8 A. PBS (control); 8B. temozolomide-cabergoline mixture; 8 C. Free temozolomide; 8 D. Free cabergoline; 8E. FOL-DUAL-DLs; 8 F. FOL-DUAL-ILs; 8 G. UN-DUAL-DLs,).

Data availability statement

All data generated or analysed during this study are included in this submitted article. The raw data shall be made available upon request to the corresponding author.