Comparative study of microscale and macroscale technique for encapsulation of Calotropis gigantea extract in metal-conjugated nanomatrices for invasive ductal carcinoma

The encapsulation of plant extract in nanomatrices has limitations due to its adhesion to walls, size control, high cost and long durations that results in low yield. Macroscale and microscale level techniques for development of micro/nanoparticles may impact the encapsulation of plant extract. This study aimed to evaluate the relative efficiency of microscale and macroscale techniques for encapsulation of plant extract, which is not compared yet. Keeping this in view, encapsulation of Calotropis gigantea leaves extract (CaG) was attained in silver-conjugated poliglusam nanomatrices (POL/Ag) to induce apoptosis in invasive ductal carcinoma (IDC) cells. The ethanolic CaG extract was prepared using percolation method and characterized by chemical tests for its active phytochemical compounds. The droplet-based microfluidic system was utilized as microscale encapsulation technique for CaG in nanomatrices at two different aqueous to oil flow rate ratios 1.0:1.5, and 1.0:3.0. Moreover, conventional batch system was utilized as macroscale encapsulation technique consisted of hot plate magnetic stirrer. The prepared nanomatrices were analysed for antioxidant activity using DPPH test and for cytotoxicity analysis using MCF-7 cells. The characteristic peaks of UV–Vis, FTIR and XRD spectrum confirmed the synthesis of CaG(POL/Ag) by both the encapsulation methods. However, microfluidic system was found to be more expedient because of attaining small and uniform sized silver nanoparticles (92 ± 19 nm) at high flow rate and achieving high encapsulation efficiency (80.25%) as compared to the conventional batch method (52.5%). CaG(POL/Ag) nanomatrices found to have significant antioxidant activity (p = 0.0014) against DPPH radical scavenging activity. The CaG(POL/Ag) of the smallest sized formulated by the microfluidic system has also shown the highest cytotoxicity (90%) as compared to batch method (70%) at 80 µg/mL. Our results indicate that the microscale technique using microfluidic system is a more efficient method to formulate size-controlled CaG(POL/Ag) nanomatrices and achieve high encapsulation of plant extract. Additionally, CaG(Pol/Ag) was found to be an efficient new combination for inducing potent (p < 0.0001) apoptosis in IDC cells. Therefore, CaG(Pol/Ag) can be further tested as an anti-cancer agent for in-vivo experiments.

Experimental setups for synthesis and encapsulation.The Pol/Ag was prepared by the conventional batch method and microfluidic systems in order to compare the encapsulation of CaG using the macroscale and microscale techniques, respectively.The experimental setup is illustrated in Fig. 1.
Macroscale encapsulation using conventional batch setup.The experimental setup of conventional batch method consisted of hot plate magnetic stirrer to prepare the CaG loaded poliglusam-silver nanomatrices (CaG-(Pol/Ag)) at macroscale 34 with few modifications as shown in Fig. 1A.The 1.5% of poliglusam (prepared in 2% of acetic acid solution) was subjected to heating and magnetic stirring at 95 °C and 120 rpm, respectively.A 4 mM of AgNO 3 solution was added drop wise into poliglusam solution in ratio 2:1 (poliglusam : AgNO 3 ) and left for 2 h of stirring.For encapsulation of CaG, 10 mg/mL of CaG was mixed into the poliglusam solution and AgNO 3 was added drop by drop.After collection of nanomatrices the 10% of NaOH was added drop wise in volumetric ratio of 0.1:1; NaOH: AgNO 3 .These samples were washed three times with deionized water %yield = We Ws X100 ) by centrifugation at 10,000 rpm for 30 min each time and lyophilized for storage at room temperature and characterization.
Microscale encapsulation using microfluidic system.The microfluidic system was used for synthesis of CaG-(Pol/Ag) at microscale which is shown in Fig. 1B.Two syringe pumps were used for dispensing of precise flow rates which are controlled by automated computer system.The poly-methyl methacrylate (PMMA) microchip developed previously in our lab 35 having T junction was used for the microemulsion formation.The straight channel carries the continuous phase (canola oil) and perpendicular channel carries the dispersed phase (poliglusam, CaG and AgNO 3 reaction mixture) shown in Fig. 1C.The dispersed phase contains 1.5% of poliglusam, 4 mM of AgNO 3 and 10 mg/mL of CaG.First of all the dispersed phase is passed through the polytetrafluoroethylene (PTFE) microreactor immersed in water bath to heat the solution up to 90 °C31 .The homogenous and stable microemulsions (water-in-oil) were formed by controlling the flow rates of both the phases.The total flow rate (TFR, mL/h) ratio of aqueous: oil was set as 0.4: 0.6 (F1) and 1.8: 5.4 (F2) and relative flow rate (RFR) ratio of 1:1.5 (F1), and 1:3 (F2).The samples were collected and 10% of NaOH was added to them (0.1:1, NaOH: AgNO 3 ) for acceleration of reduction of AgNO 3 .The collected samples were washed and stored.In order to compare both methods with equal parameters, the stirring time set for batch system was 2 h which equals to the time taken by microfluidic system to collect equal volume of nanomaterial suspension.
Determination of encapsulation efficiency.The percentage encapsulation efficiency (%EE) of Pol/Ag nanomatrices synthesized by microfluidic system and conventional batch method was calculated by measuring the free drug present in samples.For this, the CaG extract from Pol/Ag nanomaterial was separated using 10,000 rpm centrifugation for 30 min and the amount of free drug in supernatant was calculated using UV-Vis spectrophotometer at 340 nm 31 .The %EE was calculated by following formula.
where Tp is total CaG used in encapsulation and Fp is free CaG detached from Pol/Ag.

Characterization of nanomaterial.
The physical characteristics, morphology and size of particles were characterized by scanning electron microscope (SEM), X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS) and ultraviolet visible (UV-Vis) spectroscopy.

Total antioxidant capacity (TAC).
The TAC of CaG and CaG-loaded nanomaterial was evaluated by 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) solution test by using the reported protocol 32 .0.1 mM of DPPH solution was prepared in ethanol and immediately absorbance was taken at 517 nm for control reading using 96 well plate spectrophotometer (Thermo Fisher scientific).The different concentrations, 40, 60, 80, 100, 120 and 160 μg/ mL of ascorbic acid, CaG, and CaG-(Pol/Ag) nanomatrices were prepared in DMSO, and DPPH solution was added in ratio of 1:3; DPPH: antioxidant.The mixtures were shaken vigorously and placed in dark for 30 min at room temperature.The absorbance of each sample was measured at 517 nm.Here, the ascorbic acid was used as standard.
Anti-cancer activity.The CaG-(Pol/Ag) nanomatrices produced from batch and microfluidic methods were tested against the IDC cells (MCF-7) by MTT assay 31 .The 96 well plate was prepared by seeding MCF-7 (1X10 7 cells/well) cells and cultured in RPMI media for 24 h at 37 °C.The different formulations of nanomaterial with different concentrations were added into the wells and further incubated for 24 h.Afterwards, the media was removed and MTT dye (5 mg/ mL) was added into each cell, incubated for 2 h and DMSO was added to solubilize the formazan crystals.The absorbance of each cell was taken at 570 nm using the micro-plate reader.the percentage (%) cell viability was calculated using the following formula.
where, the abs(sample) and abs (control) are the absorbance of sample and control respectively.

Statistical data analysis.
The statistical data analysis was performed using GraphPad prism v.9.IC50 values were calculated and Dunnett's multiple comparisons test was applied.Two-way ANOVA was also accessed.The threshold values were set as 0.05 and sstatistical significance was established when *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.The nanomaterial and droplet size were measured by ImageJ software.The Mean and standard deviation (SD) were calculated by Graph Pad prism v.9

Results
Plant extract phytochemical analysis.The CaG extract was prepared using the ethanol as solvent by percolation method.The 50 g of dried leaves of CaG has given 9.8 g of ethanolic extract and the yield of 19.6%.This extract was found to be positive for terpenoids, alkaloids, flavonoids and phenols as indicated by reddishbrown layer at interface, orange, yellow and green colour respectively.Those findings are in accordance with previous reported studies 9,10,12 In another research, the ethanolic extract of C. gigantea has 46.75 mg of flavonoid and 33.71 mg of phenolic content 14 .The previous reported studies from HPLC and GCMS data also confirms www.nature.com/scientificreports/ of silver nanoparticles 40 .The silver nanoparticles synthesized by microfluidic system has relatively more absorbance with narrow UV-Vis spectrum, which justify the narrow size distribution of nanoparticles synthesized by microfluidic system.The CaG has peak at 340 nm which is representing the effective encapsulation of CaG in nanomaterial.Furthermore, the peak around 280 nm shows the presence of poliglusam 20 .Altogether these peaks verify the formation of nanomaterial consisted of CaG loaded in poliglusam and silver nanoparticles 31 .The UVvis spectrophotometry of CaG(Pol/Ag) prepared via the microfluidic system was performed after eight months to check the stability of the nanomatrices.A peak around 400 nm was observed which confirms the presence and stability of AgNPs as shown in Fig. 2C.
% EE of composites.The nanomatrices synthesized by microfluidic system were found to have % EE of 80.25%, while batch system has 52.5%.This shows that microfluidic system is relatively a more competent method to successfully encapsulate the high percentage of CaG extract in nanomaterial.This might be because of rapid mixing in microemulsions in the microfluidic chip 31,35,41 .Recently, Aftab et al., has achieved the 77.125 ± 6.9% EE of Calotropis procera in chitosan silver nanoparticles using the microfluidic system 31 , while we have not found any other similar study.However, other encapsulation techniques of micro and macro encapsulation has also been reported with many limitations 16,25 as discussed in Sect. 1.

FTIR spectrum.
The absorbance of IR by various functional groups of poliglusam, CaG and Pol/Ag prepared by both methods is shown in Fig. 3.It can be noted that CaG(Pol/Ag) has been successfully prepared by both methods.The shifting of pure poliglusam peaks in Pol/Ag nanomatrices may be because of formation of nanomatrices 31,42 .The broadening and elevation of peak around 3280 cm -1 shown in Fig. 3A,B as compared to Fig. 3C, and disappearance of poliglusam peak at 1639.78 cm -1 is indicating the role of hydroxyl and amino groups in stabilization of silver ions 23,34 .The CaG has displayed (Fig. 3D) the absorption peaks at various intensity which represents the presence of different functional groups existing in it.The absorbance at 1304 cm -1 , 1277 cm -1 and 1085 cm -1 confirming the presence of primary and secondary alcohols, flavonoids, and tertiary alcohol, respectively 43 .These peaks justify the bioactive compounds detected in a forementioned qualitative phytochemical analysis, which plays an important role as antioxidants.www.nature.com/scientificreports/POL/Ag prepared by microfluidic system and batch method has broad peak around 20°, along with other peaks which indicates the presence of poliglusam and natural product.For microfluidic system the appearance of Brag reflections at 2 θ = 34.48°,43.44° and 62° and for batch method at 2θ = 35.84°,42.88° and 62.8° are clearly indicating the synthesis of silver nanoparticles 42,44 .The nanomatrices (Fig. 4C,D) have a small diffraction as compared to free natural product (Fig. 4B), which is showing the crystalline form of natural product present in the composites 45 .

XRD analysis.
SEM analysis for size and structural morphology.The microfluidic system was utilised to tune the nanomatrices size at two different flow rates i.e.F1 and F2 as shown in Fig. 5.The SEM was to visualise the nanomatrices for size and morphology shown in Fig. 6A-H.

Size analysis.
It can be noticed that with the increase in flow rates (TFR and RFR) the size of microemulsion as well the size of silver nanoparticles declined.This is because with the decrease in size of microemulsion the number of nuclei decreases that are formed within each microemulsion which result in decrease in average final size of the nanomaterial 46,47 .The size of microemulsions at two different flow rates in 0.5 mm microchannel was also calculated and has been displayed in Fig. 5.At high flow rate F2, the CaG(Pol/Ag) nanomatrices of size 92 ± 19 nm were formed, while the size of nanomatrices was larger (198 ± 38 nm) at slow RFR (F1).We have also observed a narrow size distribution of nanomatrices with the increase in flow rate.All these finding is in www.nature.com/scientificreports/consistent with the previous reports [46][47][48][49] .Furthermore, the Pol/Ag nanomatrices prepared by the batch method are relatively large in size and are not evenly distributed (Fig. 6A,B).
Morphology analysis.The change in morphology of the nanomaterial captured by SEM was also observed.The SEM images has also shown the bright silver nanoparticles embedded on membrane-type-poliglusam collected from microfluidic system 42 .At high flow rate, the Pol/Ag nanomatrices are more spherical and uniform.On the other hand, the Pol/Ag nanomatrices prepared via a batch method are more irregular in shape/surface.The irregular surface of poliglusam is also not displaying the silver nanoparticles clearly.
Stability analysis.The SEM image was taken after eight months to check the stability of nonmaterices.As shown in Fig. 6G,H, after eight months the size of CaG(Pol/Ag) at flow rate F2 recorded was 119 ± 21 nm.The 27 nm increase in size was recorded which is quite negligible and also the CaG(Pol/Ag) were found to be evenly distributed.These results suggest the stability of nanomatrices.
Overall, these finding shows that microfluidic system plays an important in homogenous mixing for synthesis of narrow size distribution and smooth surface of poliglusam microcarriers.These results also indicate that the size and morphology of nanomaterial can be tuned by microfluidic system. 47,49.Yanar et al. has produced the size control liposomes 48 .While in this research we have formulated the size controlled poliglusam nanomatrices embedding silver nanoparticles conjugated with plant extract, which were not reported earlier.

Hydrodynamic size and zeta potential.
The hydrodynamic size and zeta potential of the microfluidic prepared CaG(Pol/Ag) nanomatrices was analysed using the DLS technique show in Fig. 7A-C.The zeta potential of any nanomaterial above + 30 mV is considered as highly stable and strongly cationic 50 .in this study the zeta potential of the nanomatrices was found to be 42.42 mV.Which suggests that they are highly stable and cationic.This strong cationic nature of the CaG(Pol/Ag) nanomatrices increase the endocytosis activity on the surface of negatively charged cancer cells due to electrostatic force (Fig. 7C).Therefore, this can be assumed that these nanomatrices would have ability to induce apoptosis in cancer cells to great extent.These results are consistent with earlier reported studies where the electric potential of the particles has been measured to be positively charged 51 .The average size of the nanomatrices was measured to be 178.5 nm with polydispersity index of 2.97.The size measured by the DLS is quite close to the size measured by the SEM analysis.These results indicate that CaG(Pol/Ag) nanomatrices are evenly distributed with positive charge on their surface which make them an ideal nanoagent to induce cancer cell death.

Antioxidant properties of CaG and nanomatrices.
The reported studies have found valuable phytochemical present in CaG including alkaloid, flavonoids, and phenols.Therefore, it can be used as a natural antioxidant 9,10,12 .The percentage inhibition of free radicals by CaG, CaG(POL/Ag) and standard is shown in Fig. 8.The results shows that CaG alone has adequate potential of antioxidant activity.However, when combined with Pol/Ag nanomatrices, the CaG(Pol/Ag) possess a significant antioxidant activity.
Anti-cancer effect.In this study, the IDC cells, MCF-7, were treated with the microchip-formulated (M-CaG(Pol/Ag)) nanomatrices of two different sizes, MF1-CaG(Pol/Ag) of 198 ± 36 nm and MF2-CaG(Pol/ Ag) of 92 ± 19 nm, and batch method synthesized (Ba-CaG(Pol/Ag)) nanomatrices (213 ± 73 nm).Their  Furthermore, we have also found that smallest size particles 92 ± 19 nm (MF2-CaG(Pol/Ag) at 80 µg/mL has the minimum cell viability of 10% (% cytotoxicity = 90).While Ba-CaG(Pol/Ag) nanomatrices has lowest cell viability of 30% (% cytotoxicity = 70 ) at 80 µg/mL.These results indicate that with the change in size and morphology of nanoparticles, the cell viability of the cancer cells is also affecting.A small size and uniform morphology of Pol/Ag nanomaterial has more potent anticancer efficacy [52][53][54] .However, in previous studies scientist has also preferred the 50 nm size over 10 or 20 nm 55,56 , this may be because the smallest particles may escape the cancer cells easily even before performing their average activity.While, in our study, the size of particle is around 92 nm, which is quite optimal to enter and escape the cancer cells upon their average activity 55 .
It can also be observed in this study that the antioxidant-CaG extract has shown 24% cell viability at 80 µg/ mL, however conjugating it with tuned size and morphology Pol/Ag nanomaterial has increased the anti-tumour activity 31 .The two-way ANOVA test has confirmed those finding to be statistically significant, p < 0.0001.

Conclusion
The present study reports that encapsulation of plant extract at microscale using microfluidic system is more efficient to set out high yield encapsulation efficiency as compared to encapsulation at macroscale using conventional batch method.Also, the microfluidic system provides the flexibility to control the size, shape, and polydispersity of nanomaterial by changing the flow rates of the agents.This research reports the CaG(Pol/Ag) as a new nanotherapeutic agent which is competent to induce apoptosis in MCF-2 cells.

Figure 1 .
Figure 1.The experimental setup for synthesis of CaG-loaded POL/Ag.(A) Conventional batch method using magnetic stirrer.(B) Automated microfluidic system consisted of syringe pumps and microchip of 0.5 mm channel.(C) T junction and the perpendicular and straight channels for formation of microemulsion in microchip.

Figure 2 .
Figure 2. (A) The UV-Vis spectra of Ba-CaG(Pol/Ag) and Mi-CaG(Pol/Ag) nanomatrices synthesized by batch and microfluidic system, respectively.(B) The suspension of Pol/Ag nanomaterial.(C) The UV-Vis spectra of Mi-CaG(Pol/Ag) for eight months stability analysis.

Figure 4 .
Figure 4. XRD results of CaG(POL/Ag) nanomatrices synthesize through (A) microfluidic and (B) batch method.(C) XRD of CaG extract and (D) Poliglusam solution.Arrows indicating characteristic peaks of silver nanoparticles.Asterisks * representing the various peaks of CaG extract.

Figure 5 .
Figure 5.Effect of two different flow rates on droplets and nanomaterial size/size distribution shown in table above.The microchannel and droplets with (A) F1 flow rate, and (B) F2 flow rate.

Figure 6 .
Figure 6.SEM images of CaG(Pol/Ag) nanomatrices.CaG(Pol/Ag) synthesized by batch method is shown in image (A) and histogram in (B) Synthesised by microfluidic system at different flow rates are shown in images (C) and (E) and their respective histograms in (D) and (F).Here flow rate (mL/h) in ratio of aqueous: oil phase is RFR that is F1 = 1:1.5, and F2 = 1: 3. G and H is the SEM image of F2 CaG(Pol/Ag) eight months aged.

Figure 7 .
Figure 7.The DLS analysis.(A) Zeta potential of CaG(Pol/Ag) nanomatrices.(B) Hydrodynamic size calculated via cumulative intensity distribution.(C) The diagrammatic representation of electrostatic interaction between CaG(Pol/Ag) nanomatrices and cancer cells aiding the endocytosis.