Conjugation of bisphosphonates with scorpion 2 venom enhanced apoptotic activities in 3 pancreatic cancer cells

45 Abstract: 46 Background: In recent times, prevalence and the incidence of pancreatic cancer (PC) have increased exponentially. 47 Among of the major issues of PC are the asymptotic manifestation that delays diagnosis, and scarcity of therapeutic 48 means. 49 Methodology: To provide an effective therapeutic approach, current study was executed, in which an optimized 50 alendronate sodium (ALS) and scorpion venom (SV) nanoconjugate (ALS-SV nanoconjugate) was selected via experimental 51 design applying size and zeta potential as the selected factors. Additionally, efficacy against PANC1 cells was determined by 52 analysis of IC 50 , cell cycle, annexin V staining, Bcl-2, Bax, p53 , etc. 53 Results: The particle size of the optimized nanoconjugate was found to be 28.4± 1.2 nm and the zeta potential to be 54 +26.2± 0.8 mV. The cytotoxicity in terms of IC 50 ALS-SV nanoconjugate was found better (2.73 ± 0.2 µg/mL) than ALS 55 alone (22.3 ± 0.2 µg/mL) and SV alone (6.23 ± 0.4 µg/mL). Besides, outcomes of cell cycle analysis exhibited maximum 56 efficacy for nanoconjugate in the G2-M phase. Annexin V staining, Bax, Bcl-2, p53 , and caspase 3 estimation also showed 57 superior apoptotic activity for ALS-SV nanoconjugate. However, as compared to ALS alone and SV alone, nanoconjugate 58 showed elevated expression levels of TNF-α. The apoptotic activity was enhanced by the ALS-SV nanoconjugate as 59 confirmed by MMP analysis. 60 Conclusion: Accordingly, the current study revealed an enhancement in the anti-PC effects of the ALS-SV nanoconjugate 61 that is considered as a novel approach against PC. 62 63


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Pancreatic cancer (PC) is among the major causes of death globally. As per the published report PC is the 160 Caspase 3 estimation 161 A colorimetric assay kit (BioVision, Milpitas, CA, USA) was used for the estimation of caspase 3 level in 162 treated PANC1 cells with various samples. For this purpose, PANC1 cells were cultured (3×106 cells/well) and 163 treated with various samples such as normal saline, ALS alone, SV alone, and ALS-SV nanoconjugate. In 164 addition, lysate buffer (ice-chilled) was used to suspend treated cells and left for incubation in ice for ten 165 minutes. After ten minutes, cells were centrifuged at 10,000×g for 1 min, and obtained samples were used 166 for the caspase-3 estimation as per the manufacturer's instructions, and a microplate reader analyzed the 167 evolved color at 405 nm [23,26,27]. The PANC1 cells were treated with ALS alone, SV alone, and ALS-SV nanoconjugate. The cell fraction was 171 used for the extraction of RNA and proceeded for the synthesis of cDNA. Primer for the Bcl-2, Bax, p53, 172 caspase 3, and TNF-α was designed by using Gene Runner software (Table 2) and the samples were 173 normalized with β actin [11,22].

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Determination of mitochondrial membrane potential (MMP) 175 MMP was investigated by ABCAM assay kit (Cambridge, UK) and PANC1 cells 5×10 3 (96 well plate) was 176 incubated for 24 h. ALS alone, SV alone and ALS-SV nanoconjugate were separately added. The resultant cell 177 mixture was kept in the dark, probe solution (tetramethylrhodamine, methyl ester) was replaced, and MMP 178 was observed using FACS Caliber, BD Bioscience flow cytometer [28,29].

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Mean ± standard deviation (SD) was the expressed values. One Way ANOVA (followed by Tukey's multiple 181 comparisons test) was utilized for statistical analysis (p-value < 0.05).

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Experimental design-based optimization and selection of ALS-SV nanoconjugate 185 As per the runs (15 runs) obtained from the software, which was produced with the help of selected 186 independent variables such as ALS: SV molar ratio, incubation time (min), and sonication time (min), various 187 ALS-SV nanoconjugates were prepared (Table 3). Next, for the selection of optimized nanoconjugate, all the 188 prepared ALS-SV nanoconjugates were characterized for particle size and zeta potential.

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Impacts of independent variables on particle size 190 The statistical analysis of variance of response data (particle size) has been presented in Table 4. The 191 software produced p-value declared the statistically significant effects of independent variables on selected 192 responses of ALS-SV nanoconjugates. The independent factor (X1: ALS: SV molar ratio) exhibited a positive 193 impact on particle size. Whereas independent factors X2 and X3 showed a comparatively shorter effect than 194 X1 on particle size. Additionally, the R2 value and the value of R2 adjusted for particle size were found to be 195 99.4201 and 98.3764, respectively.

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At the end of software-based statistical analysis of particle size data, a polynomial equation was found 197 (equation 1). The effects of multiple independent factors on particle size (dependent factor) can be 198 recognized by the software-produced polynomial equation. In the equation, the positive sign recommended 199 the positive effects of factors on the response. Exactly the same was seen in this study, where the particle their interconnected impacts on the particle size of nanoconjugates. In Figure 1.a Preto chart distinctly 208 demonstrated the positive effect of factors X1 and X2, and the negative effect of factor X3 over particle size.

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At the same time, software yielded contour plot ( Figure 2) affirmed the Preto chart, which stated remarkable 210 impacts of independent variables over particle size of nanoconjugates.

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Impacts of independent variables on zeta potential 213 The statistical analysis of variance of response data, i.e., zeta potential, has been presented in Table 2. The 214 software produced p-values exhibited the statistically significant effects of independent variables on the zeta 215 potential of ALS-SV nanoconjugates. The independent factor (X1: ALS: SV molar ratio) exhibited a negative 216 impact on particle size and zeta potential. However, simultaneously independent factors X2 and X3 showed 217 comparatively less effect than X1 on zeta potential. Additionally, the R2 value and the values of R2 adjusted 218 for zeta potential were found to be 99.3752and 98.2505, respectively.

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After software-based statistical analysis of zeta potential data, a polynomial equation was found (equation 2).

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The effects of multiple independent factors on zeta potential (dependent factor) can be confirmed by the after ALS alone treatment, followed by SV alone (36.91%) and the ALS-SV nanoconjugate (42.16%).

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Followed by SV alone (9.91%) and ALS alone (28.72%), and hence, it can be concluded that ALS-SV was 252 most active as an anticancer agent in the G2-M phase as presented in table 6.

Analysis of apoptotic activity via Annexin V analysis 254
Apoptosis is one of the decisive parameters for the evaluation of the anticancer potential of the drug. Annexin

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V is commonly used in the assessment of apoptotic activity. This is because Annexin has a great affinity for 256 the phosphatidylserine ligands located on the surface of cells and hence helps evaluate or identify the cells 257 undergoing apoptosis. In the present study, ALS-SV nanoconjugate induced significant necrosis in the PANC1 258 cells, followed by ALS alone and SV alone. Additionally, ALS-SV nanoconjugate showed enhanced late 259 apoptosis followed by SV alone and ALS alone . Moreover, ALS-SV also exhibited superior total apoptotic 260 activity followed by ALS alone and SV alone. However, ALS alone showed the highest apoptotic activity in 261 terms of early apoptosis, followed by SV alone and ALS-SV nanoconjugate ( Figure 5). Thus, the study 262 outcome showed superior apoptotic activity of ALS-SV during the late phase, and ALS showed early apoptotic 263 activity,as in table 7.

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Apoptosis is one of the decisive parameters for anticancer activity. Therefore, a balance between pro and 266 anti-apoptotic protein determines and anticancer potential of the drug. In the current study, the 267 administration of ALS-SV nanoconjugate significantly increased the expression level of pro-apoptotic proteins 268 such as Bax, caspase 3, and p53 and reduced the expression of anti-apoptotic protein Bcl-2 to ALS and SV 269 alone ( Figure 6). However, as compared to ALS alone, SV alone showed increased apoptotic activity. In recent times, prevalence, as well as the incidence of PC, has increased exponentially. The concern with PC 282 is the presence of asymptotic manifestation that often delays diagnosis that leads to tumor cells may reach [4]. Thus, in the current study, nanoconjugate of ALS and SV was fabricated, optimized, and characterized.

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The anticancer potential of the formulation was validated in the PANC1 cells by performing various apoptotic 287 parameters.

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The optimized ALS-SV nanoconjugate formula was generated from the Box-Behnken experimental design. In nanoconjugates was decreased with increment of ALS-SV molar ratio and incubation time, but at the same 298 time, with the increment of sonication time, the zeta potential was also increased. Therefore, it can be 299 concluded that the lower ALS-SV molar ratio and incubation time with increased sonication time produced the 300 desired quality of nanoconjugate, which contained minimal size with maximized zeta potential that was 301 required for better stability of prepared ALS-SV nanoconjugates.

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The imbalance between the cell cycle and death and changes in the