Impact of Aluminium phthalocyanine nanoconjugate on melanoma stem cells

Figures & data

Figure 1. Ligand exchange method, in which the AlPcS₄Cl sulphate (S) groups Bind onto PEGylated AuNP polymer chains.

Table 1. Excitation and emission parameters for fluorescence microscopy and flowcytometry.

Microscopy			Flow Cytometry
Fluorochromes/Dyes	Excitation/Emission (nm)	Emission Filters	Laser	Emission Filters
FITC	490/525	FITC	488nm	530/30 nm
PE	565/578	TRITC	488nm 532 nm 561 nm	585/42 nm
DAPI	358/461	DAPI

Figure 2. Ultraviolet–Visible spectroscopy (UV‐Vis) of AuNPs, AlPcS₄Cl and AlPcS₄Cl‐AuNP dispersed in ddH₂O, read at the spectral region between 400 and 800 nm. The absorption peaks are at 675 nm for AlPcS₄Cl and 520 nm for PEG‐AuNP‐COOH. Post conjugation, ALPcS₄Cl‐PEG‐AuNP‐COOH indicates absorption peaks at 525 nm and 675 nm.

Figure 3. Graphical representation of FTIR spectrum for PEG-AuNP-COOH, AlPcS₄Cl, and AlPcS₄Cl–PEG-AuNP-COOH conjugate.

Table 2. DLS, Zeta potential and PDL Characterisation.

Test	Z-Average DLS (nm)	Zeta Potential (mV)	Polydispersity Index (PDI)
AuNPs	24.92 ± 7.698	−14 ± 7.210	0.066
AlPcS4Cl	33.09 ± 34.652	−4.02 ± 12.903	0.189
AlPcS4Cl-AuNP	50.59 ± 34.923	−17.0 ± 2.023	0.015

The values are mean ± SE.

Table 3. Mean and standard deviation significance breakdown of Annexin V/PI outcomes.

Sets	Necrotic Cells (%)	Unstained Cells (%)	Early Apoptotic Cells (%)	Late Apoptotic Cells (%)
A375 CSC Stained	0.6 ± 0.173	84.7 ± 1.559	12,9 ± 1.674	1.9 ± 0.115
A375 CSC + Laser	0.7 ± 0.173	84.8 ± 0.981	13.3 ± 1.270	1.2 ± 0.173**
A375 CSC + AlPcS4Cl	0.7 ± 0.346	83.8 ± 1.790	14.0 ± 1.155	1.6 ± 0.231
A375 CSC + AlPcS4Cl + Laser	0.9 ± 0.173	71.3 ± 1.386***	25.3 ± 1.732***	2.5 ± 0.173***
A375 CSC + AlPcS4Cl-AuNP	0.6 ± 231	78.8 ± 4.734	19.8 ± 4.907	1.6 ± 0.0577
A375 CSC + AlPcS4Cl-AuNP + Laser	1.7 ± 0.404***	52.8 ± 4.503***	42.7 ± 5.081***	2.8 ± 0.173***

The mean and standard deviation of three separate assays run in duplicate is calculated to compute the values. ***p < 0.001 and **p < 0.01, respectively, reveal significant differences among the treatment and control groups (AlPcS₄Cl-AuNP: Aluminium (III) Phthalocyanine Chloride Tetrasulphonic Acid - Gold Nanoparticle Conjugate).

Figure 4. Annexin V/PI Cell death Analysis in Isolated A375 CSCs following PDT: the cells that received no treatment served as the control. The illustration clearly shows triggering of early apoptosis in A375 and CSCs. When compared to non-irradiated groups, this cell death mechanism enhanced in PS-AuNP conjugate groups irradiated with a laser at 673.2 nm with a fluency of 5 J/cm² (laser irradiation at 10.40 s, calculated using a power output of 75mW semiconductor diode laser).

Figure 5. 200x magnification Immunofluorescence microscopy of A375 CSCs expressing caspase 3 apoptotic proteins indirectly labelled with FITC (green) and counterstained with DAPI. (blue). untreated CSCs designated as a negative control, displaying no expression. Caspase 3 expression, on the other hand, is abundantly visible in the PS-AuNP conjugate + laser treated A375 and CSC groups.

Figure 6. 200x magnification Immunofluorescence microscopy of A375 CSCs expressing p53 apoptotic proteins indirectly labelled with FITC (green) and counterstained with DAPI (blue). untreated CSCs designated as a negative control, displaying no expression. PS-AuNP conjugate + laser treated A375 and CSC groups show p53 expression.

Figure 7. Post AlPcS₄Cl and AlPcS₄Cl-AuNP PDT, flow cytometry scatterplot analysis of p53 and caspase 3 apoptotic proteins on A375 total cell population and A375 CSC subpopulation. In AlPcS₄Cl-AuNP PDT groups, both apoptotic proteins were found to be overexpressed.

Data availability statement

The authors confirm that the data supporting the results are available in this article. Additional data are available from the corresponding author, upon reasonable request.