View of IN SEARCH OF BIOINSPIRED HYDROGELS FROM AMPHIPHILIC PEPTIDES: A TEMPLATE FOR NANOPARTICLE STABILIZATION FOR THE SUSTAINED RELEASE OF ANTICANCER DRUGS

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Vol.04,Special Issue 07, (RAISMR-2019) November 2019, Available Online: www.ajeee.co.in/index.php/AJEEE

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IN SEARCH OF BIOINSPIRED HYDROGELS FROM AMPHIPHILIC PEPTIDES: A TEMPLATE FOR NANOPARTICLE STABILIZATION FOR THE SUSTAINED RELEASE OF

ANTICANCER DRUGS

1Radha Rani Mehra, ²Anita DuttKonar

1,2Department of Chemistry, Rajiv Gandhi Technological University, Airport Bypass Road, Gandhi nagar, Bhopal

Abstract: The discovery of stimuli-responsive hydrogels with tunable functionality have gained significant impetus in the last decades. In accordance to this drift, herein our effort lies to modulate a series of amphiphilic peptides of general formula Me-(CH2)14-CO-NH-(X)- CH-(CH2)-4(Y)-Ph-COOH, where (X = L, Y = H, Hydrogelator I ; X = D, Y = H, Hydrogelator II

; X = L, Y = OH, Hydrogelator III ; that rigidifies water (pH:13) at room temperature irrespective of their configuration at the C-terminal centres. As determined by various spectroscopic and microscopic techniques the hydrogelators manifest β─sheet conformation and nanofibrous morphology at supramolecular level. As observed visually and confirmed by Dynamic Scanning Calorimetry (DSC) and rheological measurement the hydrogels exhibit thermo reversibility. This thermo responsive behaviour have been exploited to study the injectibility of the peptides. Additionally these hydrogels were found to be resistant towards the proteolytic enzyme proteinase K and its biocompatibility was tested using dose dependant cell viability studies employing MTT assay. To evaluate the potentiality of the hydrogelators in drug delivery we synthesized hydrogel nanoparticles (HNPs) employing the concept of self-assembly utilizing non-covalent interactions. Remarkably, these HNPs displayed an unprecedented ability to release the anticancer drugs 5-Fluoro uracil /Doxorubicin at physiological pH and room temperaturedepending on their chemical structure, molecular weight and hydrophobicity. Overall our research holds promise for utilizing this new type of peptide amphiphile based hydrogels for future drug delivery applications.

Keywords: proteolytically stable, self healing, hydrogel nano particles, drug delivery.

1. INTRODUCTION

The principle of self-assembly involves the construction of complex architectures from biological building blocks which exhibit diversified applications in drug delivery, tissue engineering, electronic devices etc.^1–4 The building blocks that have been extensively known for the generation of novel structures are nucleic acids, phospholipids and large peptidyl buildingblocks.^5–8 Like large oligopeptides, short amino acid derivatives and peptides can also self-assemble into various nano structures as well as nano scale ordered hydrogels.^9–18

Low molecular weight hydrogelators (LMOHGs) composed of amino acids and short peptides represent a promising approach to drug delivery pathways.¹⁹This is because of the fact that these molecules are of biological origin and possess non-toxic behavior. They can form specific secondary, tertiary and quaternary structures which provide unique opportunities for the design of nano materials that does not exist with traditional organic molecules.²⁰

Peptide based hydrogel nano particles (HNPs) have gained momentum in recent years as a promising nano particulate drug delivery system.^21–24 This is due to a combination of two different characteristics: (a) the hydrophilicity and extremely high water content of the hydrogels and (b) the nano particle which is exclusively small in size. The amino acid/peptidehydrogel nano particles (HNPs) can be modulated rationally by controlling the hierarchical self-assembly process which includes the non-involvement of any potentially hazardous chemicals such as cross-linkers that may affect their biocompatibility.^24–28 Moreover, their synthesis procedure is very simple and in vivo degrad ability is non-toxic due to the fact that they are composed of simple eco friendly amino acids. To date, although significant examples have been documented in the literature, the majority of them either involve synthetic and natural polymers or longer peptides as building blocks for drug delivery systems.^29–41 The exploration of hydrogel nano particles from short peptides/simple aromatic amino acid derivatives, where the concept of self- assembly has been explicitly exploited, remains in the rudimentary stage.⁴²

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Figure 1 Chemical structures of Peptide I- III

As a part of the investigation, herein we report a series of amphiphilic peptides of general formula Me-(CH2)14-CO-NH-(X)-CH-(CH2)-4(Y)-Ph-COOH, where (X = L, Y = H, Hydrogelator I ; X = D, Y = H, Hydrogelator II ; X = L, Y = OH, Hydrogelator III ; that rigidifies water at room temperature irrespective of their configuration at the C-terminal centres. As determined by various spectroscopic and microscopic techniques the hydrogelators manifest sheet conformation and nanofibrous morphology at supramolecular level. As observed visually and confirmed by rheological measurement the hydrogels exhibit thermo reversibility. This thermo responsive behaviour have been exploited to study the injectibility of the peptides. Additionally these hydrogels were found to be resistant towards the proteolytic enzyme proteinase K and its biocompatibility was tested using dose dependant cell viability studies employing MTT assay. To evaluate the potentiality of the hydrogelators in drug delivery we synthesized hydrogel nanoparticles (HNPs) employing the concept of self-assembly utilizing non-covalent interactions.

Remarkably, these HNPs displayed an unprecedented ability to release the anticancer drugs 5-Fluoro uracil /Doxorubicin at physiological pH and room temperaturedepending on their chemical structure, molecular weight and hydrophobicity.

2. EXPERIMENTAL PART

2.1 Synthesis of Hydrogelator I-III

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2.1.1 Scheme 1: Synthesis strategy of the Amphiphilic Hydrogels

The Hydrogelators Palmitic acidLF-OHand Palmitic acid DF-OH were synthesized using conventional solution phase methodology, with racemization free techniques. All the intermediates obtained were checked for purity by thin layer chromatography (TLC) on silica gel. The final derivatives were purified by column chromatography using silica gel (100-200 mesh) as the stationary phase and ethyl acetate and petroleum ether mixture as the eluent. The derivatives we have to characterize by NMR, IR spectroscopy and mass spectrometry.

2.1.2 Palmitic Acid LF-OMe: The Phe-Ome obtained from its hydrochloride (3.156gm, 14.63 mmol) was added to an ice-cold solution of Palmitic Acid (1.5 gm, 5.8495 mmol) in 10 ml of DMF. Then DCC (1.205 gm, 7.0194 mmol: 1, 3-dicyclohexylcarbodiimide) was added to the cooled mixture, which was stirred for 18 hours in an ice cold condition. The progress of the reaction was monitored by TLC. The residue was taken into ethyl acetate and the DCU was filtered off. The organic layer was washed with 2 M HCl (3 × 100), 1 M sodium carbonate (3 × 100 ml) and brine (2 × 100 ml), dried over anhydrous sodium sulphate and evaporated in vacuum to obtain a white solid material. The crude peptide was used without further purification.

Yield: 3.59625 gm, (70%, 8.6122 mmol)

2.1.3 Hydrogelator I: Compound 1 (2.0 g, 4.79122 mmol) was dissolved in methanol (28 mL) and 2N NaOH was added drop wise to the solution. The progress of the reaction was monitered by TLC. After completion of the reaction, the methanol was evaporated. The residue obtained was diluted with water and washed with diethyl ether. The aqueous layer was cooled, neutralized with 2 N HCl and extracted with ethyl acetate. The solvent was evaporated in vacuo to give a white solid.

Yield: 2.046 gm, (5.07 mmol 65%); m.p. 57-60⁰C LC-MS: 404.5 [M-H]⁺; MS (calculated) m/z: 403.59 [M]⁺; FT-IR: 940, 1296, 1434, 1465, 1542, 1644, 1703, 2851, 2918, 3405, 3455, 3566, 3595, 3623 cm^-1; 1H NMR (d6-DMSO, ppm): 12.17 (1H,br,COOH of Phe), 8.04 (1H,d, J=8Hz, NH of Phe (2)),7.25- 7.13 (5H, m, Aromatic H’s of Phe ),4.37-4.43 ( 1H,m,C^α H Of Phe (2)), 3.001-3.047 (1H, m,C^β H Of Phe(2) ) , 2.834-2.775 ( 1H, m, C^β H Of Phe(2)), 2.170-2.133 (2 H,m, C^αH’s of Palmitic acid(1) ), 1.435-1.470 (2H, m, C^β H’s of Palmitic Acid(1)), 1.211 (br,10 H, 12- methylene H’s of Palmitic Acid (1)),845-.8411 (3H, m,CH3,-H’s

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of Palmitic Acid);¹³C NMR (d6-DMSO, ppm): 175.136, 173.611, 172.832, 138.318, 129.729, 128.799, 126.728, 53.812, 37.222, 35.722, 34.376, 31.781, 29.508, 29.397, 29.290, 29.197, 29.039, 28.928, 25.655 24.968, 22.576, 14.402.

2.1.4 Hydrogelator II: Hydrogelator II was prepared using the same synthetic procedure as that for Hydrogelator I.

Yield: 2.737gm, (6.78125mmol 78%); m.p. 56-58⁰C LC-MS: 404.50 [M-H]⁺; MS (calculated) m/z: 403.59 [M]⁺; FT-IR: 940, 1297, 1434, 1465, 1543, 1644, 1704, 2659, 2851, 2919, 3296, 3665 cm^-1; 1H NMR (d6-DMSO, ppm): 12.03 (1H,br,COOH Of Phe), 8.04 (1H,d, J=8Hz, NH of Phe) 7.25- 7.16 (5H, m,Aromatic H’s of Phe (2) ), 4.37-4.42 ( 1H,m,Cα H Of Phe (2)), 3-.05 (1H, m,Cβ H Of Phe(2)), 2.77-2.83 ( 2H, m, C^β H Of Phe(2)), 2.137-2.73(2 H,m, C^αH’s of Palmitic acid(1) ), 1.437-1.472(2H, m, C^β H’s of Palmitic Acid(1)), 1.26-1-21 (10 H, m, 12- methylene H’s of Palmitic Acid (1)), 814-.848 (3H, m,CH3,-H’s of Palmitic Acid)

; ¹³C NMR (d6-DMSO, ppm): 134.926, 173.817, 172.446, 138.382, 129.515, 128.891, 128.891, 34.130, 31.775, 29.519, 29.387, 29.498, 29.223, 29.189, 29.030, 24.968, 22.573, 14.15.

2.1.5 Hydrogelator III: Hydrogelator III was prepared using the same synthetic procedure as that of Hydrogelator I.

Yield: 2.3562gm, (5.6164 mmol 72 %); . LC-MS: 420.49[M-H]⁺; MS (calculated) m/z: 419.59 [M]⁺; m.p. 55-57⁰CFT-IR: 941, 1228, 1297, 1465, 1530, 1648, 1701, 1745, 2851, 2919, 3473 cm^-1; 1H NMR (d6-DMSO, ppm): 11.926 (1H,br,COOH Of Tyr), 9.164 (1H,br, NH of Tyr ), 6.95(2H, d,J=8Hz), 4.30-4.37 ( 1H,m,Cα H Of Tyr), 3.55-3.55 (OH of Tyr), 2.83-2.91 ( 1H, m, C^β H Of Tyr), 2.69-2.83 (1 H,m, C^β H’s of Tyr), 2.14-2.17(2H, m, C^β H’s of Palmitic Acid), 1.436-1.472 (2H, m, C^β H’s of Palmitic Acid), 1.21-1.26 (10 H, 12- methylene H’s of Palmitic Acid ), .847-.813 (3H, m,CH3,-H’s of Palmitic Acid); ¹³C NMR (d6-DMSO, ppm):

174.9, 130.4, 127.7, 115.5, 34.2, 31.7, 29.5, 29.4, 29.3, 29.2, 29.1, 25.01, 22.58, 14.4.

2.2 Preparation of the Hydrogel. For preparation of the hydrogels, 5 mg of each of the hydrogelators were dissolved in 1ml of phosphate buffer in glass vials by heating on a hot plate to obtain a homogenous mixture. These solutions were then slowly cooled to room temperature (20⁰C) and kept undisturbed for the gelation studies. The formation of the hydrogel was confirmed by the inverted vial method.

2.3 Rheology: Rheological measurements were carried out at 25^oC using a Rheoplus MCR302 (Anton paar) rotational rheometer with parallel plate geometry and the data were processed using start rheometer software. For the oscillatory shear measurements, a parallel top plate with a 25 mm diameter and 1.0 mm gap distance were used. A frequency sweep experiment was performed from 0.11 to 100 rad/s at constant strain of 1 %. The viscoelastic properties of the hydrogels were measured by measuring the storage modulus G′ and loss modulus G′′. Hydrogels (1 ml) were transferred on a rheometer plate by using a microspatula and kept hydrated using a solvent trap. A stainless steel parallel plate (diameter: 25 mm) was used to sandwich the hydrogels with TruGap(0.5 mm). The dynamic strain sweep experiment was performed to determine the reason of deformation of hydrogels in which linear viscoelasticity is valid. The exact strains for hydrogel materials were determined by linear viscoelastic regime at a constant frequency of 10 rad/s. The mechanical strengths of the hydrogels were determined by frequency sweep experiment. In the aforementioned measurement the graphs were plotted against function 0.11 to 100 rad/s at constant strain of 1 %.

2.4 Field Emission scanning electron microscopic study (FESEM).The FE-SEM experiments were performed on a JEOL scanning electron microscope (JEOL JSM - 6700F).

For the morphological measurements small amounts of hydrogels of concentrations (1X,2X,3X and 4X-mgc)obtained were placed on a glass cover slip. The hydrogels were dried first in air and then in vaccum and coated with gold.

2.5 Proteolytic stability assay: To determine the resistance of our designed peptides against proteolysis, the Hydrogelators were incubated with proteinase K from Engyodontium album. Five hundred µM of the peptides were first dissolved in 50 mM Tris

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buffer, containing 5 mM CaCl2, at pH 8. The peptide solutions were then incubated with 500 units of proteinase K for 24 hours at room temperature.

2.6 MTT Assay: Cytotoxicity of the Hydrogelators were determined through MTT (methyl thiazolyl tetrazolium) colorimetric assay, which measures cell metabolic activity through the conversion of tetrazolium dye to insoluble purple formazan crystals as per the method described. The dipeptides were dissolved in DMSO stock solutions followed by making the dilutions (with DMSO <0.1%) directly using cell culture media. No precipitation or aggregates were observed even on storage up to 72 hours at 37⁰C. The cells were seeded on flat-bottom 96 well plates at a density of 4000-5000 cells/well. After 24 h, the cells were drugged with serial dilutions (0, 0.1, 0.3, 1, 3, 10, 30, 100 µM) for each of the compound in triplicate. After 72 h of incubation, MTT dye (4mg/ml) was added to all wells and incubated at 37 °C for an additional 4 h. The media was carefully discarded after this incubation period and formazan crystals were dissolved in 100 µL of DMSO in each well for 15 minutes. Absorbance was measured at a wavelength of 570 nm using a DTX 880 multimode detector (Beckman Coulter Life, Indianapolis IN, USA).The raw data was analyzed and plotted using GraphPad Prismv7.02.Student’s t-test was used to analyze all the data.

2.7 Nano particle Characterization. Nanoparticle size diameter and surface charge were measured using Malvern Zetasizer, with 4mW 633 He-Ne Laser, (DTS version 4.10, Malvern, U. K.) with appropriate viscosity and refractive index settings. The temperature was maintained at 25^oC during the measurement.

2.8 In vitro drug release of curcumin, 5-Fluoro-uracil and Doxorubicin from HNPs. The in vitro release profiles of the drugs 5-flurouracil, curcumin and doxorubicin from the drug loaded HNPs were determined using a dialysis membrane previously soaked for 24 hrs in the dissolution membrane and stretched around at one end of a tube. The drug loaded formulations were placed on pretreated membrane immersed into 22 mL of solvent (for curcumin, enzyme free SIM in ethanol (50% v/v) and for 5-fluorouracil and doxorubicin, phosphate buffer of pH-7.5, was used) at room temperature and magnetically stirred at 250 rpm. At selected time intervals, aliquots were withdrawn from the release medium and replaced with same amount of solvent(for curcumin, enzyme free SIM in ethanol (50% v/v) and for 5-flurouracil and doxorubicin, phosphate buffer of pH-7.5). The samples were analyzed in triplicates using a UV-spectrophotometer with wavelengths of λ 273nm for 5- fluorouracil and 495nm for doxorubicin. The percentage of cumulative drug release was plotted against time to obtain the release curves.

3. RESULTS AND DISCUSSIONS

3.1 Preparation and characterization of the hydrogels

From the literature documentation, it is evident that weak interactions like (H-bonding, hydrophobic and p-pinteractions) play a vital role in gelation. Keeping this view in mind, we have synthesized a series of molecules comprising of Palmitic acid at the N-terminus coupled to various aromatic amino acids of diversified configuration.

These Hydrogelators I-III undergo hydrogelation with a minimum gelation concentration of 0.05% w/v without any sonication at room temperature. At first the hydrogelators were dissolved in a drop of DMSO under mild heating conditions and allowed to cool. To this, the required amount of phosphate buffer (pH: 7.5) was added to make the volume up to 1 mL. It was allowed to stand at room temperature for 8–10 min to obtain an opaque gel. The formation of the gel was confirmed using an inverted test tube method.

The gels are stable over a period of six months. Sol–gel transition temperatures (Tgel) for both the hydrogelators I and II (% w/v) were plotted against different concentrations of hydrogelators(Figure. 2). These plots show that the Tgel values of the aromatic amino acid derivatives increase with an increase in the concentration (% w/v) of the hydrogelator until the plateau region is reached. This plateau region indicates that the formation of the hydrogel network has reached the saturation limit. No further addition of any molecule is capable of increasing the gel melting temperature (Tgel value).

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6 12 18

40 48 56

Hydrogelator III

Tgel (

o

C )

Concentration (mg/ml)

Hydrogelator I

Hydrogelator II

Figure 2 The change in the Tgel profile of hydrogelators with respect to concentration While investigating thermal stability, we observed that the hydrogels initially melt to form a transparent solution upon the application of heat. However, after the withdrawal of heat, this sol reverts back to the gel. Typically, this is a characteristic of thermo reversible in jectible polymers.

Therefore, to confirm our observation, we repeated the experiment several times, followed by injecting the solutions to and from in a syringe. Interestingly, during each case, we not only deciphered the transformation of gel to sol and vice versa on application / withdrawl of heat respectively, but each time this transition period was decreased.

We anticipate that this reversible nature might be attributed to the high mechanical integrity which in turn imparts high elasticity, inject ability, and self-healing nature in the hydrogels.

Figure 3 Thermo reversible property of Hydrogelator II

To understand the mechanism governing the self-assembly propensities of the Hydrogelators I – III FTIR measurements were carried out. The FTIRspectra of the xerogels obtained show a major band at 3300–3340 cm_1, a characteristic feature of hydrogen bonded NH stretching. Both xerogels of the derivatives I and II further exhibited peaks in the region 1696, 1694 cm_1 (amide I) and 1538, 1536 cm_1 (amide II), respectively, characteristic features of CQO stretching and NH bending frequencies, respectively. These data therefore suggest the presence of -sheet conformations in the xerogels.

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Figure 4 FT-IR spectra of the dried gels of Hydrogelator I – III

In order to gain insight into the morphology of the hydrogels, a field emission scanning electron microscopy (FESEM) investigation was carried out. The FESEM images (Figure. 5) of the xerogels of exhibit flat net-like morphology.

4000 3500 3000 2500 2000 1500 1000 500

0 30 60 90

% T ra ns m itt an ce (a .u )

Wavenumber(cm^-1)

Hydrogelator I (solid) Hydrogelator I (xerogel)

3500 3000 2500 2000 1500 1000 500

40 80 120 160 200

% T ra ns m itt an ce

Wavelength (cm-¹)

Hydrogelator II(solid) Hydrogelator II(xerogel)

4000 3500 3000 2500 2000 1500 1000 500

30 60 90

Tr an sm itt an ce (a .u )

Wavenumber (cm^-1)

Hydrogelator III (solid) Hydrogelator III (xerogel)

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Figure 5 FE-SEM images of the dried gels of Hydrogelator I – III

In order to address the mechanical strength and stability of the hydrogelators, rheological studies were performed where the storage modulus G’ (elastic response) and loss modulus G’’ (viscous response) were measured in frequency sweep experiments. As is evident from Fig. 4 (left), in the lower viscoelastic region (LVR) the storage modulus G’ is higher than the loss modulus (G’’) for both of the amino acid derivatives. This observation suggests a soft ‘solid-like’ gel phase formation. Even the thermo reversible property was established by the temperature dependant measurement versus complex viscocity.

Figure 6 Rheological measurement curves of Hydrogelator I – III

The Hydrogelators I - III contain the palmitic acid residue, known to be resistant towards proteolytic cleavage.³⁵To verify this phenomena, the Hydrogelators were incubated with proteinase K as previously described. Enzymatic degradation was monitored by measuring the corresponding mass spectra after 48 hrs at regular intervals of 6hrs. The absence of the peak corresponding to Pal (256) (i.e. the degradation product), and the observance of the peptide peak corresponding to (M) ⁺(Hydrogelator I and II: 403; III: 418) after 48 hrs, affirms their resistance towards proteolysis.

Figure 7 Proteolytic Stability Data

1 10 100

10³ 10⁴ 10⁵

M od ul us (P a)

Angular Frequency (rad/sec)

G' of Hydrogelator I G'' of Hydrogelator I G' of Hydrogelator II G'' of Hydrogelator II G' of Hydrogelator III G'' of Hydrogelator III

30 40 50

10⁵ 10⁶ 10⁷

Co m pl ex v is co si ty (  )

Temperature (^oC)

Hydrogelator III

Hydrogelator II Hydrogelator I

0 20 40

90 100

Compound rema ined (%)

Time (hour)

Hydrogelator I Hydrogelator II Hydrogelator III

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150 300 450 600

30 60 90 120

Activity (%)

Concentration (M)

Hydrogelator I

IC50:294.50 M

Figure 8 MTT assay results for Hydrogelator I, II and III

Furthermore the hydrogelators did not show any profound impact on the morphology of the cell lines. Also the cells were found to remain viable when tested through MTT assay, thereby confirming the biocompatibility of the hydrogelators.

Thus, our results indicated that the hydrogelators display proteolytic stability and biocompatibility.

We developed nano particles, using our hydrogelators, to assess their applicability for drug delivery. Hydrogel nano particles were synthesized using a modified inverse emulsion process (water-in-oil) as previously described. The hydrogelators hada bimodal distribution, with an average particle size of 304.6± 47.12 nm and 21.32± 4.15 nm for Hydrogelator I, 171.6± 30.01nm and 15.71± 2.046 nm for Hydrogelator II, and136.1± 21.16 and 23.27± 4.93 for Hydrogelaor III. Moreover, a high negative zeta potential (-18.7 mV) conferred formulation stability. This negative charge is attributed to the presence of carboxylates in the derivative as well as the surfactant coating around the hydrophobic core. Our results also demonstrated that the particles have similar diameters and morphology even after six months of storage, thereby emphasizing the stability of the formulation. We hypothesize that the drug molecules would remain entrapped within the core and would be released after exposure to a stimulus.

Figure 9: Hydrogel Nanoparticle Formulation (HNP) preparation by modified Inverse Emulsion Method.

0 100 200 300 400 500 600 0

20 40 60 80 100 120

Concentration (M)

% Activity

Hydrogelator II

IC50: 352 M

0 300 600

40 60 80 100

% Activity

Concentration (  M)

Hydrogelator III

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To test the versatility of our Hydrogelators as carriers for drugs with varying properties (i.e. chemical structure, molecular weight and hydrophobicity) we chose 5-FU and doxorubicin as model drugs. The corresponding drug release profiles were monitored using methods as described earlier. ¹ The drug release profiles, as presented in Figure 6H, showed a marked dependence on the respective molecular weight of the drug candidates particularly for doxorubicin. Though the initial rate of release for 5-FU seemed faster compared to curcumin, the net cumuliative release of the respective drugs remained more or less similar for the two hydrogelators. However, the Hydrogelator-II released less drug compared to Hydrogelator-I

0 10 20 30 40 50 60 70 80

% Cumulative Drug Release

Time in Hours

Hydrogelator I With 5FU

0 10 20 30 40 50 60 70 80

Time in Hours

Hydrogelator II With 5 FU

35 40 45 50 55 60 65 70 75 80

20 30 40 50 60 70 80

Time in Hours Hydrogelor III With 5FU

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Time in Hours

Hydrogelator I With DOX

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0 10 20 30 40 50 60

Time in Hours

Hydrogelator II With DOX

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40 60 80 100 120 140

0 10 20 30 40 50 60

Time in Hours

Hydrogelator III With DOX

Figure. 10 Release Profiles of Hydrogelator I – III

Which could be attributed to their respective gel strengths. In addition 5-Fluoro Uracil binding to both Hydrogelators occurred exclusively through hydrogen bonding. In contrast, curcumin binding occurred primarily by π─πinteractions, whilst for the case of doxorubicin, both π─stacking as well as H-bonding seemed important. Hence for the latter case extensive drug matrix H-bonding could explain the slower release of drug from Hydrogelator II.

Hence our experimental findings coupled with our computational analysis indicate that our synthesized Hydrogelators can be used as potential drug carriers.

4. CONCLUSION

In summary, we have demonstrated the formation of three simple amphiphilic derivatives that display excellent hydrogelating abilities under mild conditions. The gelation properties of the hydrogel were investigated using various microscopic techniques and its strength was determined by rheological measurements.

Interestingly, these amino acid conjugate based hydrogels have been nicely tuned to prepare and stabilize hydrogel nano particles (HNPs) utilizing an inverse emulsion procedure. The method proposed here is environmentally benign, as simple amino acid conjugates have been utilized. The approach adopted is the concept of self-assembly utilizing weak interactions, unlike other polymers that require harsh conditions and complex techniques for their fabrication. To date, significant examples have been documented in the literature for synthetic and natural polymers and longer peptides/peptide amphiphiles where they have been used as building blocks for the drug delivery systems. However, to the best of our knowledge, these derivatives represent one of the very few reports of HNP formation resulting from the self assembly of simple amphiphilic derivatives, solely driven by an environmentally benign approach i.e. weak interactions, unlike other polymers that require drastic conditions and complex techniques for their fabrication. The obtained HNPs were characterized and evaluated for their size, zeta-potential and post-formulation stability. Our gelators display good entrapment efficiency and in vitro release kinetics of the model drug 5-FU. The obtained results clearly indicate that these amino acid derivative based HNPs may be suitable for use as a potential drug delivery system. However, further studies to evaluate the candidature of this novel type of amino acid conjugate based HNPs for nano medical applications are under investigation.

5. ACKNOWLEDGEMENTS

Radha Rani Mehra wishes to thank UGC (F/-17/2017-18/ RGNF-2017-18-SC-MAD- 34810) New Delhi, for financial support, Dr. Anita Dutt Konar for her valuable guidance for carrying out the research work and Dr. Sanjit Konar, IISER Bhopal, for providing facilities at IISER Bhopal.

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