Gel-Emulsion Properties of Nontoxic Nicotinic Acid-Derived Glucose

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Gel-emulsion Properties of Nontoxic Nicotinic Acid Derived Glucose Sensor. Aparna Roy, Sumita Roy, Ananya Pradhan, Sujata Maiti Choudhury, and Rati Ranjan Nayak Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b04187 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

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Industrial & Engineering Chemistry Research

Gel-emulsion Properties of Nontoxic Nicotinic Acid Derived Glucose Sensor. Aparna Roy, a Sumita Roy,*a Ananya Pradhan,b Sujata Maiti Choudhuryb and Rati Ranjan Nayakc a

Department of Chemistry and Chemical Technology, Vidyasagar University, Paschim

Medinipur-721 102, India. b

Department of Human Physiology with Community Health, Vidyasagar University, Midnapore 721

102, India c

Lipid Science and Technology Division, Indian Institute of Chemical Technology, Hyderabad

E-mail: [email protected] Abstract Amide linkage containing two nicotinic acid amphiphiles have been investigated for their gelemulsion and glucose sensing properties. In emulsions, the synthesized materials were used as stabilizers in water medium, whereas organic solvents and/or mineral oils as dispersed phase and water acts as a continuous phase. The gel-emulsions were prepared at room temperature by stirring or shaking and no heating-cooling arrangement or addition of any other co-solvents, active agents are mandatory. Rheological investigation of these amphiphiles showed mechanical stability of gel-emulsions and its viscoelasticity. Optical images confirmed the existence of network structures in gel-emulsion phase for both the amphiphiles. FE-SEM measurement suggests the morphology depends on the solvent:water composition. XRD study proposed that the arrangements of the amphiphiles in the emulsion state are different. The gel-emulsions formed by the studied amphiphiles are able to entrap and release bio-molecule, anticancer drug

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molecule and hazards chemicals. These gelator molecules are very sensitive to detect glucose molecule in aqueous solution. The sub-chronic toxicity of these synthesized molecules has been evaluated as non-toxic on the hematological and biochemical parameters of male Swiss albino mice. Introduction In recent years the most investigated research topic is molecular gels. The molecular gels are a heterogeneous preparation composed of two immiscible materials. The molecular gels are formed in various ways, by adding a small amount of active agents which self-assembled in water (hydrogel) or in organic solvents (organogel) or in oil-in-water (O/W) / water-in-oil (W/O) (gel-emulsion). When the volume fraction of dispersion phase is 0.7 or greater typically known as gel-emulsion. After several decades of research on gel-emulsion, the most accurate definition of gel-emulsion is defined as emulsions containing a dispersion phase volume fraction of 0.74 or greater.1,2 Due to non-equilibrium in nature, the properties of gel-emulsions are strongly dependent on particular methods are used to prepare gel-emulsions. For preparation of gelemulsions firstly appropriate amount of amphiphiles is dissolved in the continuous phase and the component which acts as a dispersed phase was added stepwise under continuous shaking or stirring. 3-7 The gelation instigated by shaking or stirring i.e. mechanical forces is a key stimulus in terms of applications. In recent years, Stimuli-responsive materials are showing remarkable development for its wide applications in sensors, displays, biomaterials, surface science, etc.8-11 Different factors such as pH,12 light,13 redox,14,15 and enzymatic effect

16-19

etc. are responsible

for alternation of gel properties of low-molecular-weight gelators (LMWG). Basically, gelemulsions have been extensively used in food chemistry,20,21 cosmetics,22,23 pharmaceutical,24,25 chemical industry,26,27 and for templates preparation.7,28−30 In soft matter research the study of


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gel-emulsion becomes a hot point due to its attractive nature and wide range application. Some of the gelator molecules have different sensing property due to the presence of different binding sites. Here we disclosed glucose sensing properties of our amphiphiles as glucose metabolism is a crucial factor for living organisms especially for millions of diabetic patients. Moreover, findings of glucose sensing is highly important in fermentation industry, biochemistry, and clinical diagnosis.31,32 The enzyme based detection of glucose has several limitations such as they are unstable, difficult to sterilize, they have slow sensor time lags and the process is expensive.33,34 The optical response is preferred due to its ability to detection remotely. After a long research, it has been identified that in aqueous media boronic acids are crucial ligand for detection of the glucose molecule.35-37 It is well documented that gelator molecules have capability to intercalate cell membrane by insertion into the cell membrane which changes the molecular organization of membrane that accomplish cell lysis.38 Evaluation of toxicity is the most important criterion for their uses in biological and pharmaceutical field however, toxicity of synthetic amphiphiles is still a barrier for their use in biological research. So in biological research the evaluation of the toxicity of the gelator molecules is essential. Encouraged by the literature report and to observe the enormous utility of the gel-emulsions, in this work we have demonstrated stimuli responsive gel-emulsion properties of two synthesized nicotinic acid derivatives having amide linkage in the head group named Sodium 6dodecylnicotinic alaninate (SDDNAA) and Sodium 6-dodecylnicotinic valinate (SDDNAV) (Figure 1). In our earlier publications39-41 we have reported the gel-emulsion property of some nicotinic acid and boronic acid based gelators. In this paper, the applicability of the gelemulsions formed by SDDNAA and SDDNAV has depicted for entrap and release capacity of biomolecules like vitamin B12, anticancer drug doxorubicine and hazards chemicals such as



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methylene blue, cresol red and crystal violet. The sensing ability of these two amphiphiles toward glucose molecules has been investigated. Moreover, the toxicity of these compounds has also been evaluated.

O

R = CH3, SDDNAA CH(CH3)2, SDDNAV

R -

+

O Na N H

O

N

Figure 1. Molecular structure of synthesized compound Results and Discussion Gelation property of amphiphiles SDDNAA and SDDNAV are capable to form gel-emulsions in different organic solvents, mineral oils and vegetable oils in presence of 50µl water (Table 1). We observed that both these amphiphiles form gel-emulsion at room temperature with gentle shaking by hand and remains intact as usual even after four months. A screw cap vials (i.d. 1 cm) were inverted and no downward flow of the sticky material was observed which confirmed amphiphiles form stable gel-emulsions (Figure S1 of SI). Without water as additives, these amphiphiles were unable to form gel in organic solvents, vegetable oils and mineral oils either by heating-cooling process or by agitation. It was visually observed that the transparency of the gel-emulsions decreases from lower chain aliphatic solvent to higher chain aliphatic solvents. This observation suggests formation of larger type aggregates which have sizes in between 400nm-700nm (visible light) in higher chain length aliphatic solvent. The obtained gelation parameters such as Minimum Gelation Concentration (MGC) values and “gelation number,” Ngel of the amphiphiles enlisted in Table 1. Both these amphiphiles are good gelator in eighteen organic solvents, however, SDDNAV act as better gelator compared to SDDNAA as MGCSDDNAV < MGCSDDNAA and Ngel


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SDDNAV > Ngel SDDNAA.42 This result implied that hydrophobicity of the head group facilitates the process of gelation.

Table 1. Gelation parameters of SDDNAA and SDDNAV.

Solvent

MGC (gm L-1) SDDNAA SDDNAV 0.250 0.360

Tgel  0.05 (K) SDDNAV SDDNAA SDDNAV 8506 -

Gelation number (Ngel) SDDNAA 11407

Petroleum ether (4060) 0.160 0.100 20863 35822 Pentane 0.245 0.140 11909 22360 Hexane 0.305 0.200 8536 13967 Heptane 0.230 0.180 26146 35844 Dichloromethane 0.220 0.200 21684 25591 Chloroform 0.305 0.220 12987 19317 Tetra chloromethane 0.323 0.260 11004 14667 Cyclohexane 0.353 0.210 12241 22077 Benzene 0.400 0.240 9065 16209 Toluene 0.520 0.250 6122 13662 o-Xylene 0.301 0.270 10335 12361 m-Xylene 0.297 0.250 12597 16056 Nitro benzene 0.263 0.150 9855 18540 Kerosene 0.27 0.195 5091 7564 Diesel 0.300 0.230 9198 12872 Mesitylene 0.310 0.250 11795 15692 Bromo-benzene 0.317 0.275 11956 14787 Chloro-benzene 0.345 0.258 Nut oil 0.400 0.312 Til oil 1.110 0.852 Sunflower oil 0.254 0.212 Olive oil Effect of Amphiphile Concentration and Water on Gel-emulsion

329 340 343 349 351 353 355 341 339 344 340 348 355 -

332 341 346 351 353 354 358 348 343 347 344 351 357 -

The effect of amphiphile concentration and volume of water added on gelation property were also investigated. The plots for amount of solvent gelled against amount of amphiphile (Figure



S2A) concluded that maximum amount of solvent gelled linearly increases to the concentration of the amphiphile. Figure S2B represents how the volume of water affects the gelation ability of a given amount of amphiphile. At first there was an increase in volume of solvent gelled as volume of water increases and then gradually decreases reaching a maximum which indicated a fixed amount of water is required for gelation for a given amount of amphiphile. Temperature Effect on the Gel-Emulsion In order to examine thermotropic nature and thermal stability of gel-emulsions, tube-inversion technique was employed. Although the gel-emulsions are observed remains stable at ambient temperature for more than four months in a sealed test tube, they malformed to solution/dispersion above a certain temperature widely have known as gel-to-sol transition temperature (Tgel). At Tgel the gel-emulsions are transformed to sol state and the gel state reformed when cooled at room temperature with gentle shaking by hand. Table 1 listed Tgel values of SDDNAV and SDDNAA (C = 1.0 mg ml-1) which are in the range 329-358 K. The high Tgel of the gel-emulsions suggests that both the gel-emulsions are thermally stable. The data tabulated in Table 1 showed that SDDNAV has slightly higher Tgel values than SDDNAA which suggested that SDDNAV has better thermal stability than SDDNAA. Thermodynamic Parameters of the Gel-Sol Transition To evaluate different relevant thermodynamical parameters,43,44 Tgel values of the gel-emulsions were measured over a range of amphiphile concentrations and ln[gelator] was plotted as a function of 1/Tgel of the gelator in a particular solvent to obtain the gel-sol phase transition curve (Figure S3).45,46 The chemical potential (µ), the enthalpy (∆Hgel →sol), entropy (∆Sgel →sol) and free energy (∆Ggel

→sol)

of gel to sol transition process of the gel-emulsions formed by the

amphiphiles were estimated following the equations described in SI and the obtained results are


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included in Table 2. From the data listed in Table 2, it can be concluded that negative value of entropic change of gel-sol transition process is compensated by high negative value of enthalpy. The ∆Hgel

→sol

values of the gel-emulsions of the amphiphiles are negative which indicated

formation of intermolecular hydrogen bonds during the gelation process. The greater negative values of ∆Ggel

→sol

implied that the gelation processes are spontaneous. However, the more

negative ∆Ggel →sol value of SDDNAA than SDDNAV concluded that gelation process is more favorable for SDDNAA. Table 2. Thermodynamic parameters of the gel-emulsions formed by SDDNAA and SDDNAV at 298 K. Properties ∆H gel →sol (kJ mol-1) ∆S gel →sol (J K-1 mol-1) ∆G gel →sol (kJ mol-1)

SDDNAA -70.95 -97.73 -100.08

SDDNAV -60.46 -98.86 -89.92

Rheological Studies Rheology experiment was performed to get the idea of mechanical stability and viscoelastic properties of the gel-emulsions. Two rheological parameters storage modulus G′ and loss modulus G″ which are related with energy storage and energy loss respectively were monitored as a function of shear stress (σ) and frequency at room temperature. Figure 2A,B shows that at fixed frequency (1Hz) both moduli decreases sharply above a critical stress value representing a fractional breakup of 3D-network structure of the gel-emulsion. The yield stress (σy) values for both the amphiphiles were obtained from critical stress point. The yield stress values are 10.15 Pa for the gel-emulsions formed by both the amphiphiles. This observation suggests that gel networks in both the gel-emulsions are stable. Reference to the Figure 2 exposed that for both the



samples, G′ is higher than G″ at low stress region, which proposed that gel-emulsions under study are elastic materials.

50

40

40

A

30

G' G''

20 10

B

30 20 10

0 20 40 60 80 100

0 1 2 3 4 5

1000

G' G''

0

0

20 40 60 80 100

Shear Strees (Pa)

0

10

20

30

Frequency (Hz)

Shear stress (Pa)

12000

32 40

5

10

15

'

G '' G

3000 0

24

E

16

G', G'' (Pa)

0

G', G'' (Pa)

D

9000

'

''

C 2000

0

0

6000

G' G''

G', G'' (Pa)

50

G', G'' (Pa)

G', G'' (Pa)

3000

G , G (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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G' G''

8 0

0

10

20

30

40

Frequency (Hz)

50

30

F

20 10

G' G''

0

20

30 40 50 60 0 Temperature ( C)

70

20 30 40 50 60 70 0 Temperature ( C)

Figure 2. Change of G' and G'' with respect to σ of gel-emulsions of (A) SDDNAA and (B) SDDNAV; with respect to frequency of (C) SDDNAA and (D) SDDNAV; with respect to temperature of (E) SDDNAA and (F) SDDNAV. Figure 2C,D shows the results obtained from frequency sweep measurements. In the studied frequency range 0 to 50 Hz, G′ and G″ does not cut to each other for both amphiphiles which indicates no crossover point is present. This implicated long lifetime of the gel-emulsions. For both gel-emulsions dumping factors (DF = G″/G′)7 are lower than 1, which implied solid-like viscoelastic nature of the three dimensional networks in the gel-emulsion state. This result strongly suggests that gel-emulsions are quite stable towards applied external forces during frequency range 0 to 50 Hz. Figures 2E,F are the plots representing the data obtained from


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temperature sweep experiments. This experiment was carried out to detect the gel transition temperature in emulsified state of these amphiphiles. At Tgel, a state change from semi solid to liquid like viscoelastic state takes place, which indicates the presence of G' = G" region. Both the plots do not show presence of gel-sol transition temperature up to ~343 K. This phenomenon confirmed formation of stable and strong conformation between the gelator molecules through intermolecular H-bonding among the amide linkages. Morphology of Gel-Emulsions Optical microscopy and FE-SEM techniques were used to obtain the internal morphology of studied gel-emulsions.

B

A

C 50µm

50µm 50µm

D

E

F

50µm

50µm

50µm

Figure 3. Optical images of gel-emulsions of SDDNAA in (A) benzene and of SDDNAV in (B) m- xylene; Fluorescence micrographs of gel-emulsions using Rhodamin B as probe molecule of SDDNAA in (C) hexane and of SDDNAV in (D) toluene and using Fluoroscein as probe molecule of SDDNAA in (E) xylene and of SDDNAV in (F) cyclohexane.



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Optical Microscopy: The optical micrographs represent in Figure 3A,B confirmed the existence of network structures in gel-emulsion phase for both the amphiphiles. The networks are connected through different sizes of spheres with average diameter of the compartments ~20-250 µm. Fluorescence micrographs using Rhodamin B (Figure 3C,D) and Fluoroscein (Figure 3E,F) confirmed presence of similar types of network structures in gel-emulsion state formed by gelator molecules. FESEM:

B

A 2 µm

D

C 2 µm

10 µm

2 µm

E

F

G

H

1 µm

3 µm

1 µm

2 µm

Figure 4. FESEM images of freeze dried gel in cyclohexane of SDDNAA at composition water:solvent = 1:45 (A, B) and 1:90 (C, D) and of SDDNAV at composition water:solvent = 1:80 (E, F) and 1:150 (G, H). The actual morphological features of the gel-emulsions were investigated using field emission scanning electron microscopy (FESEM) technique after freeze drying of these emulsified samples. For both the amphiphiles SDDNAA and SDDNAV, the SEM pictures were visualized at two different compositions of solvents keeping the fixed concentration of the respective amphiphile. The obtained pictures are shown in Figure 4. The SEM micrographs of SDDNAA at


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a composition 1:45 of water:benzene showed micrometer fibers of width ~50-250 nm as well as emulsified morphology having three-dimensional (3-D) network structure (Figure 4A,B). At higher solvent content (1:90) the freeze dried sample of SDDNAA revealed emulsified morphology containing small spheres of the sizes ~100-650 nm (Figure 4C,D). However, SDDNAV at lower solvent content (water:benzene = 1:80) exhibited emulsification as well as scoop like aggregates made of fibers (Figure 4E,F). At higher solvent content (1:150) both emulsified structures and bundle of fibers (Figure 4G,H) were observed. X-ray Diffraction Studies (XRD) XRD measurements were carried out to investigate molecular packing in gel phase of selfassembled amphiphiles SDDNAA and SDDNAV. Figure S4 represents XRD peaks in small angle regions of xerogel. Table 3 summarizes the peak positions (2θ) values, corresponding planes and inter-planar distances (d). The XRD spectrum of SDDNAA shows existence of only one type of conformation (cisoid, 100%). But presence of two types of repeated peaks in XRD spectrum of SDDNAV confirmed presence of two types of morphology. Therefore, in addition to cisoid conformation (62%), transoid conformation (38%) appears considerable amount for the bilayers of SDDNAV. This is due to the fact that the bulkiness of the head group of SDDNAV hindered the cisoid conformation and hence causes the existence of the transoid conformation of the aggregates. The arrangement of gelator molecules in cisoid and transoid conformation is shown in scheme S2. Table 3 suggests that mode of arrangement of molecular packing in “cisoid” (I) and “transoid” (II) conformation is similar. The peaks of low intensities at higher angles (Table 3) demonstrate repeated order of packing in the emulsified network. The peaks are well matched corresponding to the planes (001)I and (001)II. From these two planes for two morphologies, other planes with appropriate (hkl) values can be assigned.47



Table 3. XRD parameters of the gel-emulsions formed by SDDNAA and SDDNAV.

Amphiphiles

Solvent

SDDNAA

Toluene

SDDNAV

Cyclohexane

2θ 2.20 4.39 6.59 2.11 2.55 4.24 5.13 6.32 7.65 9.64

Dried-gel d (Å) (hkl) 40.24 (001)Ι 20.17 (002)Ι 13.44 (003)Ι 41.98 (001)Ι 34.76 (001)ΙΙ 20.91 (002)Ι 17.38 (002)ΙΙ 14.06 (003)Ι 11.33 (003)II 9.21 (004)ΙI

lc (Å) 20.72

20.74

Application of the gel-emulsion It has been already stated that 50 µl aqueous solution of both the amphiphiles form gel-emulsion towards a variety of solvents including mineral oils and vegetable oils. Optical and FE-SEM images revealed that in emulsion state network, spherical aggregates and fibrillar network structures are present. So there are many void spaces which form stencil to entrap and release of biomolecules, hazards chemicals and drug molecules. On the basis of this idea we have studied the utilization of the gel-emulsion in entrapment and release of vitamin B12, methylene blue, crystal violet, cresol red and doxorubicin. The visual insight of entrapment and release of bio molecule, hazards chemicals and doxorubicin are given in SI (Figure S5). The entrapment and release phenomena of the gel-emulsions formed by the amphiphiles were investigated spectrophotometrically using all of these probe molecules. For this measurement, six pHs in the range 2 to 7.5 were tried40 and absorbance of gel-emulsions were recorded at 550 nm, 664 nm, 433 nm, 590 nm and 480 nm for vitamin B12, methylene blue, cresol red, crystal violet and doxorubicine respectively. At pH = 2, the entrapped molecules


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releases very fast from the network structures whereas there was no release in other pHs (pH = 2.5, 3.0, 3.5, 5.5 and 7.5) for the amphiphiles. Therefore, both the amphiphiles show stability at hydrolytic pH 7.5 and it can be predicted that the pKa value of the gel-emulsions is in between the range of pH 2.0-2.5. The pKa of gel-emulsions were determined by performing the same experiment from pH 2.0 to pH 2.5 with an increase of pH 0.1.40 It was observed that gel network does not break at pH 2.1 for SDDNAA and at pH 2.3 for SDDNAV gel-emulsions. Figure 5 represents the release profiles of the gel-emulsions as a function of time at pH 2 and 7.5. Assuming the release phenomena follow the first-order kinetics, k and half-time (t½) of the processes were calculated and are tabulated in Table S1. The results suggest that release rate of entrapped biomolecule and cresol red is less for SDDNAV; whereas for other materials (crystal violet, methylene blue and doxorubicin) the release rate of SDDNAV is higher than SDDNAA.

1.0

1.0

Methylene Blue Cresol Red Crystal Violet Doxorubicine Vitamin B12

0.8 0.6

Release (%)

Release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

pH = 7.5

0.4 0.2

0.8

B

0.6

Methylene Blue Cresol Red Crystal Violet Doxorubicine Vitamin B12

0.4 0.2

pH = 7.5

0.0

0.0 0

4

8

12

16

Time (Sec)

20

24

5

10 15 20 25 30 35 40

Time (sec)

Figure 5. Time dependent percentage release of (A) SDDNAA and (B) SDDNAV of vitamin B12 at two different pHs. To inspect the release phenomena of doxorubicin, fluorescence microscopic technique was used. For this purpose the gel-emulsions of these amphiphiles were prepared in 50 µL 3 mM doxorubicin. At first, 3D-network structures with entrapped doxorubicin were observed under



the fluorescence microscope (Figure S6). Then 20 µL buffer solution of pH=2 was added to the prepared gel-emulsions of the amphiphiles and again the microscopic images were captured. The images (Figure S6) clearly showed release of doxorubicin upon destruction of network structures. Glucose sensor The glucose sensor property of two synthesized gelators was also evaluated. The fluorescence spectra were obtained by excitation of two gelators-fluorophores at 268 nm and the emission spectra were recorded in between 300 nm to 600 nm. The fluorescence emission intensity of 5×10-5 M gelators (SDDNAA and SDDNAV) was quenched in presence of glucose molecules.

30

Fluorescence Intensity

Fluorescence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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A 20

10

0 530

540

550

Wavelength (nm)

100

B 50

0

530

540

550

Wavelength (nm)

Figure 6. Fluorescence emission spectra of (A) SDDNAA and (B) SDDNAV in presence of Dglucose. Figure 6 shows the glucose concentration was increased from 1 to 13 mM keeping SDDNAA and SDDNAV concentration fixed (5×10-5 M), the intensity of the fluorescence spectra was decreased continuously. Scheme S1 represents the binding sites of glucose molecule with the gelator molecule. The standard deviation of blank measurements was calculated by measuring the emission intensity of the gelator molecules 10 times in absence of glucose molecules. A linear quenching of fluorescence emission intensity was observed upon increase in glucose


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concentration from 1 to 13 mM. The detection limit was determined by using following relation:48 detection limit =3σbi/m

(1)

where σbi is standard deviation of blank measurements, m is slope of the plot I0/I vs. glucose concentration (Figure S7). The respective plots for determination of standard deviation of the amphiphiles are given in SI (Figure S7). The measured detection limit was found 0.34 mM for both the gelator molecules. Toxicity Study A molecule having low toxicity towards animal cell line is very essential for novel applications like gene delivery. To measure the potential toxicological hazards, the sub-chronic repeated dose toxicity study is generally conducted for 4 weeks. Sub-chronic repeated dose toxicity study in mice Animal maintenance and treatment Twenty healthy male Swiss albino mice of 18-22 g body weight were taken for toxicity test. These animals were randomly divided into four sub-groups having five animals each and were maintained under standard laboratory conditions for 7 days before commencing the study.49 This experiment was permitted by the Institutional Animal Ethical Committee (IAEC). A sub-chronic repetitive dose toxicity study was conducted for 28 days (4 weeks) as per the OECD 407 guidelines.50At first, 1000 mg kg-1day-1 dose was administered for the induction of toxic effects in mice and the mice were unaffected and no lethality or severe suffering was observed.51 Thereafter, 500 and 250 mg kg-1day-1 dose levels were selected to demonstrate the dose related response and no adverse effects were observed even at a dose 250 mg kg-1day-1. After this, aqueous solutions of SDDNAV and SDDNAA were injected intraperitoneally at the dose levels



of 250 (group I), 500 (group II) and 1000 mg kg-1 (group III) for 28 days daily by sterile syringe52 and the control mice (group IV) were given only water. At the beginning and last day of the treatment, weights of all animals were taken with an interval of seven days. Throughout the study period, any observed toxicity or mortality were recorded. On 29th day, all animals were sacrificed. Heart, spleen, kidneys and livers were carefully separated and weights were taken (Table S2 and S3). Body weight and clinical signs: Changing in body weight is the indicator of adverse effects for the foreign material and if >10% loss of initial body weight is seen, it will be concluded as statistically significant toxicity.53,54 Individual organ weight is also being a critical indicator of physiological and pathological states of animals. The relative organ weight is also an important parameter to endorse whether the organ weight are due to injury or not. A toxicant can alter metabolic reactions by which the primary organs i.e. heart, liver, kidney, spleen may be affected.55 For our compounds, it was observed that body weight was unaffected in treated animals against the controlled species (Table S2, S3). No clinical sign of reactions or any macroscopic changes was seen after treatment. No significant differences in absolute or relative organ weights were observed in liver, spleen, heart and kidney between the treated and control groups. From these results, primarily it can be considered that tested compounds are nearly non-toxic. Hematology and clinical evaluation To evaluate the toxicity of any compound, hematological parameters are most sensitive factors in animals as well as in humans and a blood profile usually provides important information on the reaction of body for any stress or damage.56, 57 Therefore, hematological and clinical study was done to examine the toxicity of the studied amphiphiles in mice blood. As shown in the Table


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S2, S3 we could not observe any significant dose dependent toxic hematological effects in mice after 28 days for the tested amphiphiles. The hematological parameters of exposed animals remained in the normal range as compared to the control animals. The biochemical parameters like serum glutamate oxaloacetate transaminase (SGOT) and serum gluamate pyruvate transaminase (SGPT) are two key serum biomarker enzymes generally evaluated to measure any toxic effects of the drug molecule on liver.58 In our study, no significant changes in SGOT and SGPT levels (Figure 7) were observed in mice.

c

SDDNAA 1000mg/kg body weight

SDDNAV 1000mg/kg body weight





0

Control

60

20

c

Blood glucose

80

40

*

SDDNAA 1000 mg/kg body weight

0.0

SDDNAV 1000 mg/kg body weight

0.5


1.0


1.5


2.0

Control

mg/dl

2.5



3.0

100

mg/dl







Control

mg/dl

10 5 0


120 *

Serum Urea

Serum Creatinine

3.5

c

45 40 35 30 25 20 15


0


12

4

c



16

8

4.0

SGPT

20

IU/lit



0


2


4

Control

8


10


12

6

24

SGOT

14

Control

16

IU/lit

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


c

Figure7. SGOT, SGPT, Serum creatinine, Serum urea and Blood glucose level after treatment of SDDNAV and SDDNAA for 28 days in Swiss albino mice. Results are expressed as Mean ± SEM. Analysis is done by one way ANOVA. Comparison was done between control groups versus all other groups. (*indicates p

Gel-Emulsion Properties of Nontoxic Nicotinic Acid-Derived Glucose

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