Nanoparticles Obtained by Using Different Gelation Solution

Nanoparticles Obtained by Using Different Gelation Solution3. RESULTS AND DISCUSSION3.1 surface of nanoparticles obtained by development unlike gelation solution3.1.1 sizing of distinct alginate- ground nanoparticles at fixed enzyme and Polyoxyethylene sorbitan mono-oleate (surfactant) concentrationVarious alginate base nanoparticles were prep atomic number 18d by victimization different gelling solutions as given in method . The coat of nanoparticles determined by DLS is given in table-1.Table-1 Showing size and intensity of nanoparticles at single concentration of surfactant used and enzyme immobilizedS. No.Surfactant Conc. (mM)Enzyme Conc. (mg/mL)Cross-linking agentPeak size, diam (nm)Day 0Intensity (%)Peak size, diam (nm)Day 3Intensity (%)1.8.2032.5BaCl289.43790.697.8374.42.CaCl287.88394.3161.6543.SrCl269.19375.291.38624.NiCl2421.833 ascorbic acid537.675.5Size was measured on the resembling day as the preparation of alginate based nanoparticles and there was uniformity in size distri neverthelession of the peak diam which is shown in figures-1, 2, 3 and 4. fancy 1 Size distribution of Ba-alginate nanoparticles A) on same day and B) after cardinal eld.Figure 2 Size distribution of Ca-alginate nanoparticles A) on same day and B) after three days.Figure 3 Size distribution of Sr-alginate nanoparticles A) on same day and B) after three days.Figure 4 Size distribution of Ni-alginate nanoparticles A) on same day and B) after three days.It could be seen that when size was determined on the same day (Day 0), uniformity was observed in the peak diameter. However, when size was determined after 3 days from the development of nanoparticles, the size was found to increase and the distribution was random. This happens due to Oswald ripening.3.1.2 Size of different alginate based nanoparticles at varying surfactant concentrationDifferent alginate based nanoparticles were developed without enzyme immobilization at different concentrations of surfactant rangi ng from below critical micelle concentration value to its double as given in Method . The size of nanoparticles is depicted in table-2.Table-2 Showing size and intensity of nanoparticles at different concentrations of surfactant used (no enzyme immobilized)S. No.Surfactant Concentration (mM)Cross-linking agentPeak size, diameter (nm)Day 0Intensity (%)1.0.006BaCl2CaCl2SrCl2NiCl2104.570.01165.913553.757.96179.52.0.012BaCl2CaCl2SrCl2NiCl2384.5150463.7193.594.31001001003.0.024BaCl2CaCl2SrCl2NiCl2339.271.0349.65127.552.850.969.362.8It has been observed from the table that as the concentration of surfactant increases, the size and shape incur more regular. Below the critical micelle concentration of the surfactant, uneven and irregular do particles were formed. This observation is validated by the size termination of the nanoparticles using dynamic infirm scattering at different concentrations of the surfactant as shown in figures 5, 6, 7 and 8.Figure 5 Size distribution of Ba-alginat e nanoparticles A) at below CMC (0.006mM) B) at CMC (0.012mM) and C) above CMC (0.024mM) of the surfactant. D) fleck of the peaks of alginate nanoparticles obtained against three different concentrations of surfactant used.Figure 6 Size distribution of Ca-alginate nanoparticles A) at below CMC (0.006mM) B) at CMC (0.012mM) and C) above CMC (0.024mM) of the surfactant. D) Plot of the peaks of alginate nanoparticles obtained against three different concentrations of surfactant used.Figure 7 Size distribution of Sr-alginate nanoparticles A) at below CMC (0.006mM) B) at CMC (0.012mM) and C) above CMC (0.024mM) of the surfactant. D) Plot of the peaks of alginate nanoparticles obtained against three different concentrations of surfactant used.Figure 8 Size distribution of Ni-alginate nanoparticles A) at below CMC (0.006mM) B) at CMC (0.012mM) and C) above CMC (0.024mM) of the surfactant. D) Plot of the peaks of alginate nanoparticles obtained against three different concentrations of su rfactant used.3.1.3 Size of different alginate-based nanoparticles at varying enzyme concentrations but fixed surfactant concentrationNanoparticles of various sizes and shapes were made by varying the concentrations of enzyme which was immobilized in the alginate matrix as described in Method. The different sizes obtained against differently immobilized enzyme concentrations are shown in table-3.Table-3 Showing size and intensity of nanoparticles at different concentrations of enzyme immobilized against a constant surfactant concentrationS. No.Surfactant Conc. (mM)Enzyme Conc. (mg/mL)Cross-linking agentPeak size, diameter (nm)Day 0Intensity (%)1.8.2031BaCl2CaCl2SrCl2NiCl279.11110.766.4861.7953.377.25158.42.2.5BaCl2CaCl2SrCl2NiCl289.43787.88369.193421.83390.694.375.21003.5BaCl2CaCl2SrCl2NiCl265.78146.570.09138.251.183.343.558.24.7.5BaCl2CaCl2SrCl2NiCl281.18218.883.9165.8454.691.463.280.6Figures 9, 10, 11 and 12 show the changing size of the nanoparticles with change in the concentrat ion of enzyme immobilized in alginate matrix. A comparative graphical plot has alike been incorporated to display the change in size against varying enzyme concentration for each of the cross-linking agents.Figure 9 Size distribution of Ba-alginate nanoparticles A) at 1mg/mL B) at 2.5mg/mL C) at 5mg/mL and D) at 7.5mg/mL of enzyme concentration encapsulated in alginate nano-beads. E) Plot for the variation of peak size against changing enzyme concentration.Figure 10 Size distribution of Ca-alginate nanoparticles A) at 1mg/mL B) at 2.5mg/mL C) at 5mg/mL and D) at 7.5mg/mL of enzyme concentration encapsulated in alginate nano-beads. E) Plot for the variation of peak size against changing enzyme concentration.Figure 11 Size distribution of Sr-alginate nanoparticles A) at 1mg/mL B) at 2.5mg/mL C) at 5mg/mL and D) at 7.5mg/mL of enzyme concentration encapsulated in alginate nano-beads. E) Plot for the variation of peak size against changing enzyme concentration.Figure 12 Size distributi on of Ni-alginate nanoparticles A) at 1mg/mL B) at 2.5mg/mL C) at 5mg/mL and D) at 7.5mg/mL of enzyme concentration encapsulated in alginate nano-beads. E) Plot for the variation of peak size against changing enzyme concentration.From the various figures of nanoparticles, it could be seen that the average peak size of nanoparticles increase with increasing concentration of enzyme for Barium, Calcium and Strontium. However, in case of Nickel, the size is maximum at 2.5mg/mL concentration of enzyme and it decreases for high concentrations of enzyme.3.1.4 Size of different alginate-based nanoparticles at different pH for fixed enzyme and surfactant concentrationsNanoparticles of different sizes and forms were prepared by varying the pH of the buffer solution as described in Method. The peak size diameter of the nanoparticles synthesized is given in table-4.Table-4 Showing size and intensity of nanoparticles at different pH of buffer for enzyme immobilized in alginate matrix against a constant surfactant concentrationS. No.Surfactant Conc. (mM)Enzyme Conc. (mg/mL)pH of BufferCross-linking agentPeak size, diameter (nm)Day 0Intensity (%)1.8.2032.55.36BaCl2CaCl2SrCl2NiCl2178.9256.1292349.184.688.971.91002.7.04BaCl2CaCl2SrCl2NiCl289.43787.88369.193421.83390.694.375.21003.10BaCl2CaCl2SrCl2NiCl2254.9608.2205.149684.857.678.7100Figure 13 Size distribution of Ba-alginate nanoparticles A) at pH 5.36 B) at pH 7.04 and C) at pH 10 of the buffer of alginate matrix. D) Plot shows the variation of peak size against changing pH.Figure 14 Size distribution of Ca-alginate nanoparticles A) at pH 5.36 B) at pH 7.04 and C) at pH 10 of the buffer of alginate matrix. D) Plot shows the variation of peak size against changing pH.Figure 15 Size distribution of Sr-alginate nanoparticles A) at pH 5.36 B) at pH 7.04 and C) at pH 10 of the buffer of alginate matrix. D) Plot shows the variation of peak size against changing pH.Figure 16 Size distribution of Ni-alginate nanoparticles A) at pH 5.36 B) at pH 7.04 and C) at pH 10 of the buffer of alginate matrix. D) Plot shows the variation of peak size against changing pH.From figures 13 and 14, it can be clearly seen that size of the nanoparticles is the smallest at pH 7 and largest at pH 10 for BaCl2 and CaCl2. In case of figure 15, size is smallest at pH 7 but largest at pH 5.36 for SrCl2. However, in case of figure 16, size increases in ascending order from pH 5.36 to pH 10 for NiCl2.3.2 Determination of membrane structure of the nanoparticles using infrared spectroscopyThe characteristic bands for different regions of sodium alginate and its cross with the nanoparticles developed through Method using BaCl2, CaCl2, NiCl2 and SrCl2 solutions as cross-linking agents are shown in figure 17.Figure 17 FT-IR results of alginate nanoparticles showing intensity bandsFrom figure 17, it is clear that all peaks have shifted downfield. This results in stretching of the bonds between various functional groups and so bond space of increases.Spectroscopic analyses of the alginate-based nanoparticles were based on three distinctive regions of intensity and frequency. The spectroscopic peaks obtained from the graph and their relative assignment to various regions or vibrations or stretching are given in table-5.Table-5 FT-IR Transmittance bands (cm-1) of alginate-based nanoparticlesBariumNanoparticleCalciumNanoparticleStrontiumNanoparticleNickelNanoparticleAssignment72088690810241038107211181154128613781464161016261734234628522922295634363448720886908964994102410721118115212841378140814521464160016081690172823462852292229563434345067071883288689290695210941250129413501378145014601638172423442362285229222954344236763690377038063822390667671090295210181154129813181350140614381460148215481642196223462852292029603010343038063904 (CO), (CC), (COH) (CO), (CCO), (CC) (CO), s (CC) (COC), (OH) (OH), (CH), (CH), (CH).s (COO)Amide IIAmide Is (CH2)a (CH2) stretching bending twisting wagging s symmetric a asymmetri cThe carbohydrate region is present between frequencies 1200-800 cm-1 as is shown in figure 19. span of (C O) + (C C) + (C O H) vibrations give the carbohydrate region. The mean peak for Barium and Calcium was observed at 1072 cm-1 while for strontium it was observed at 1094cm-1.The overall FT-IR spectra of the different alginate-based nanoparticles are shown in figure 18.The protein region is present between 1700-1480 with bands centered draw near 1640 cm-1.Asymmetric stretching bands of carboxylate group (a COO) were observed near 1600 cm-1 for the various nanoparticles and symmetric stretching band of carboxylate group were centered near 1462 cm-1. In infrared spectra the methylene groups show asymmetric stretching (a CH2) near 2922 cm-1 and symmetric stretching (sCH2) near 2852 cm-1. OH and NH stretching (3000-3600 cm-1) with peaks at 3436 cm-1 (for Ba), 3434 cm-1 (for Ca), 3442 cm-1 (for Sr) and 3430 cm-1 (for Ni).N.B. Results of FT-IR spectra of D-series nanoparticles are awaited.Figure 18 FT-IR spectra of A) Ca-alginate nanoparticles B) Ba-alginate nanoparticles C) Sr-alginate nanoparticles and D) Ni-alginate nanoparticlesFigure 19 FT-IR spectra for carbohydrate region (1200-800cm-1) of A) Ca-alginate nanoparticles B) Ba-alginate nanoparticles C) Sr-alginate nanoparticles and D) Ni-alginate nanoparticlesFigure 20 FT-IR spectra for protein region (1700-1480cm-1) asymmetric and symmetric COO stretching of A) Ca-alginate nanoparticles B) Ba-alginate nanoparticles C) Sr-alginate nanoparticles and D) Ni-alginate nanoparticles3.3 Determination of shape and size of alginate-nanoparticles using SEMDLS method is not a perfect technique for the determination of size of nanoparticles. So SEM studies are undertaken to have accuracy in size measurement.Figure 21 SEM picture of A) Ba-alginate nanoparticles B) Ca-alginate nanoparticles C) Sr-alginate nanoparticles and D) Ni-alginate nanoparticles (same scale for all images).For SEM-imaging of alginate based n anoparticles prepared using various geling conditions, the samples were gold coated as described in Method.Average size of barium-alginate nanoparticles was 86.8 nm (diameter) and the shape of the beads formed after enzyme encapsulation were spherical. Spherical shaped beads were also formed in case of calcium-alginate nanoparticles with average size of 51.4 nm (diameter). Strontium-alginate nanoparticles also had spherical shaped bead formation with average diameter of 45.3 nm. In case of nickel-alginate nanoparticles, the majority of the particles were rod-shaped with average height of the rods being 310.8 nm. Also, some minor beads were formed having spherical shape and average diameter of 102.3 nm.3.4 Measurement of UV-visible spectraThe UV-visible spectral determination of absorbance of the alginate based nanoparticles was determined indoors 200-400nm baseline range.Figure 22 UV-visible spectra of Ba-alginate nanoparticles A) At CMC (w/o enzyme) B) At double CMC (w/o enzyme) C ) At 1mg/mL enzyme concentration D) At 2.5mg/mL enzyme concentration E) At 5mg/mL enzyme concentration and F) At 7.5mg/mL enzyme concentration.Figure 22 UV-visible spectra of Ca-alginate nanoparticles A) At CMC (w/o enzyme) B) At double CMC (w/o enzyme) C) At 1mg/mL enzyme concentration D) At 2.5mg/mL enzyme concentration E) At 5mg/mL enzyme concentration and F) At 7.5mg/mL enzyme concentration.Figure 23 UV-visible spectra of Sr-alginate nanoparticles A) At CMC (w/o enzyme) B) At double CMC (w/o enzyme) C) At 1mg/mL enzyme concentration D) At 2.5mg/mL enzyme concentration E) At 5mg/mL enzyme concentration and F) At 7.5mg/mL enzyme concentration.Figure 24 UV-visible spectra of Ni-alginate nanoparticles A) At CMC (w/o enzyme) B) At double CMC (w/o enzyme) C) At 1mg/mL enzyme concentration D) At 2.5mg/mL enzyme concentration E) At 5mg/mL enzyme concentration and F) At 7.5mg/mL enzyme concentration.From the spectral figures 21, 22, 23 and 24 it can be clearly seen that the MAX for the d ifferent alginate-nanoparticles is around 235nm. It can also be clearly seen that the protein content at 280nm increases with increase in the concentration of enzyme immobilized in the alginate matrix. The spectra of the alginate nanoparticles show peaks and stretching only indoors the UV range of 200-330 nm of the spectra and then the absorbance becomes constant. The nanoparticles which were made without the immobilization of enzyme at CMC and double CMC of Polyoxyethylene sorbitan mono-oleate had the least absorbance at 280 nm in all the different types of nanoparticles depicting the fact that no enzyme was encapsulated within them.3.4.1 Variation of O.D. with protein contentFigure 25 O.D. variation plot against varying protein content at 280nm for A) Ba-alginate nanoparticles B) Ca-alginate nanoparticles C) Sr-alginate nanoparticles and D) Ni-alginate nanoparticles.From figure 24, it can be seen that Nickel-alginate nanoparticles have the highest amount of protein immobilized wi thin the matrix. While, the protein immobilized in Barium, Calcium and Strontium alginate nanoparticles are observed to have the similar amounts of protein immobilized in them.3.4.2 UV-visible spectra for d-block element-based alginate nanoparticlesThe overlay of alginate nanoparticles developed from d-block elements is shown in figure 26.Figure 26 UV-visible spectra overlay for A) Na-alginate B) Co-based alginate nanoparticles C) Cu-based alginate nanoparticles D) Fe (II)-based alginate nanoparticles E) Fe (III)-based alginate nanoparticles F) Mn-based alginate nanoparticles G) Ni-based alginate nanoparticles and H) Zn-based alginate nanoparticles with no enzyme encapsulation.Figure 27 (a) UV-visible spectra for A) Na-alginate B) Co-based alginate nanoparticles C) Cu-based alginate nanoparticles D) Fe (II)-based alginate nanoparticlesFigure 27 (b) UV-visible spectra for E) Fe (III)-based alginate nanoparticles F) Mn-based alginate nanoparticles G) Ni-based alginate nanoparticles an d H) Zn-based alginate nanoparticles with no enzyme encapsulation.