INTRODUCTION:

Hydrogels are well known polymeric system that not dissolves in aqueous medium, however, they swell in water, as they are capable of absorbing large amounts of water or biological fluids, this is due to the thermodynamic property of this matrix^1,2. The history of hydrogels is back to many decades, the first appearance for the scientific term “Hydrogel” was in 1894. Particularly, in 1900 the “Hydrogel” was used for a colloidal gel of inorganic salts. In the following years, the researchers were focused on the utilizing the hydrogels in ophthalmic and drug delivery applications³. Around 1998, the artificial proteins of hydrogel were discovered to have the ability for self-assembling. The self-assembly peptides are formed from coiled or β-sheet ordered structure, when the oligomerization of the helical ends was balanced with the swelling of water-soluble central moiety, then the self-assembly peptides are formed⁴.

Tailoring the amino acids sequence in the peptide, facilitates changing the chemical and mechanical properties of the peptides⁵. To illustrate, the physicochemical and mechanical features of the hydrogels, can be modified through changing the sites of amino acids, enabling to get various chemical interactions and conjugations.

Over the past decades, there were several trials and techniques, for developing the self-assemble peptides with the nanostructure and β-sheet^6,7 . Alignment of peptides as parallel or anti-parallel, form β-sheet bilayers and fibrils by inter and intra molecular hydrogen bonds as well as electrostatic interactions^6,8.

Notably, the self-assembly peptides forming β-sheet structure. In other words, the hydrophilic amino acids can be seen on the one side (face) of the β-sheet, while the hydrophobic moiety on the other face⁹. Recently, hydrogels have been applied in many fields, e.g., food additives, pharmaceuticals and drug delivery systems¹⁰ .

Self-assembled hydrogels are generated from hydrophobic interactions. The amino acid sequences of the hydrogels are responded to the PH and temperature changes^{9 .}The possibility of changing the amino acids during the peptide design, leading to the flexibility of developing a plethora of peptide sequences, that is ending with various peptide structure. On the other hand, this peptide sequences designing, make plausible to get the self-assembly fibril structure of peptide¹¹.

The goal of this study is 1- to prolong the injectable drug doxorubicin, Figure 1 release from hydrogel system over time, by varying the number of the lysine residues. 2-Detect the effect of different PH on the peptide structure.3-Determine the gelation at different concentrations.4-Visualize the topography of the hydrogels.5-Study the rheological profile of hydrogels. Our trial herein is to load doxorubicin within hydrogel to monitor the drug release at target tumour cells.

Doxorubicin is an anthracycline antibiotic is used as a drug model to be incorporated with the hydrogel. The cornerstone of cancer therapy is a cytotoxic drug^12,13. Doxorubicin is the most common cytotoxic drug which is clinically approved against solid tumours. Recently, anthracyclines particularly doxorubicin have been widely used. Despite this class of chemotherapy have some limitations, such as cardio toxicity and narrow therapeutic window.

Figure 1: Chemical structure of doxorubicin HCl. ⁵

Doxorubicin as is come from anthracycline family, its well-known to be given by intravenous injection. The anthraquinone moiety of doxorubicin which holds positive charge is participated in aromatic interactions and intercated within double strands of DNA, inhibiting topoisomerase-II (Top-II), DNA alkylation, and DNA cross-linking, inhibiting DNA helicase activity¹⁴.The aromatic and electrostatic interactions might be responsible to stabilize the forces between amphiphilic, acidic β-sheet peptides and doxorubicin⁵. There were previous attempts to control the doxorubicin release from hydrogels, Hiroshi Saito and co-workers have been studied the release of doxorubicin from Polyethylene Glycol PEG hydrogels, that have Schiff Base Linkages. Additionally, doxorubicin release from the hydrogel decreased. Thus, the doxorubicin release profile from hydrogels dependent on the PH¹⁵. However, there is a main limitation for using PEG-based systems, which is the biodegradable resistance, therefore, this system designed carefully to prevent the need for removal the device by surgery¹⁶ . In biomedical applications the hydrogels have been employed as carriers for drug delivery. The benefit of this conjugation (drug with hydrogels) is to monitor the sustained release of the drug over time¹⁷. Several attempts to control doxorubicin release have been reported that the drug release rate inversely proportional to the degree of conjugation¹⁸.

A new approach is used to deliver anticancer agent is by nanofiber peptides. The self-assembling peptides form nanostructures under certain physiological conditions. In general, assembling of the peptides can be performed via temperature, electrolytes and PH. Different hierarchical structures for the peptide arrangements, such as tapes, ribbons, fibrils and nanofibers that related to the self-assembly peptides. These peptides with the self-assembly and amphiphilic surfactant properties, having the ability to form cylindrical or fibril geometries rather than micelles, resulting in nanostructures⁶. Peptide amphiphile with nanofiber character has a paramount importance to deliver the drug¹⁹. Amino acids containing different numbers of lysine residues, naming (P2, P3 and P4) have been used in this project. These numbers of lysine are correlated to the rate of drug release from the peptides. Amphiphile peptides are self-assembling gelators, that have been proved their magic in the cancer therapies and other bioactive applications. These peptides agrregates as self-assemble cylinder, which is divided into three regions: 1-Hydrophobic region, 2-B-sheet amino acids and 3-Charged hydrophilic region²⁰. The structural properties and the concentrations of the peptides are evaluated to relate these features with the drug release from the gel texture, at PH≈5²¹. These amphiphile peptides designed with a free amine (-NH2) at the N-terminus. These molecules are self-assembled by π-π interactions, hydrogen bonding and Van der Waals interactions. The Micro or Nano network structure of the amphiphiles contains number of cavities, where the water molecules are immobilized at certain conditions to form hydrogels²².It is noticeable that designing the hydrogels with nanofiber structure and carbon nanotubes will boost the mechanical and strength properties, involving π-π interactions. These features of the nanofiber can emphasise the role of the hydrogels in drug delivery, because this will prolong the drug release from the hydrogel matrix and take time to diffuse out. Nanostructure of the hydrogel can be designed and formed fibrous network, achieving from these self-assembled peptides⁴.

The amphiphilic peptides are hybrids that formed from peptide head and alkyl chain, contain the hydrophobic and hydrophilic amino acids with oppositely charged amino acids, these charges could affect the solubility, this can be controlled through changing the PH. Different types of interactions are involved among these moieties such as aromatic interactions, electrostatic interactions, Van der Waals forces, and hydrogen bonds, forming B-sheet structure⁶ . In this study, is confirmed that the relationship between β-sheet structure particularly the number of lysine residue and the doxorubicin release from the hydrogels, ending with the major aim which is sustained the anticancer drug release from the gels to the tumour cells²³. The amphiphilic peptides exhibit a thixotropic property, shear-thinning. This means the hydrogels undergo separation of gel from the solution when subjected to strain or mechanical stress, then recovers and re-assembled on the stress removal²² .

MATERIALS AND METHODS:

Materials:

The chemicals that have been used are: the peptides P2 (FEFKFEFK), P3 (FEFKFEFKK), and P4 (KFEFKFEFKK), K=lysine, E=glutamic acid, F=phenylalanine, are purchased from Cambridge Reagents. From Emplura the Sodium Hydroxide was purchased, while the HPLC grade water was received from Thermo Fisher. Doxorubicin was supplied by Cambridge Reagents, the vortex was adapted from Manchester university. All other materials and solvents are purchased from Sigma-Aldrich and have been used as received.

Preparation of hydrogels:

Various kinds of peptides powder (P2, P3 and P4) were weighed at different concentrations (5, 10, 15, 20, and 30mg), and dissolved in 350µL of HPLC grade water. The Stuart vortex mixer SA8 was utilised to accelerate the mixing for about 50 seconds at 2500rpm, using sonic bath to check the dissolving peptides. Titration through stepwise addition of sodium hydroxide solution (0.5M NaOH), to achieve self-assembly again mixing by vortex at 50 seconds after each addition. The peptides gelated at PH from 4.5 to 5.5. Gelation or “sol-gel transition” is occurred depending on the structure and composition of the starting materials, when these materials linking together, upon exposed to external stimuli or at specific addition. The gelation process ending with increased the size of formulations and changing the solubility, whereas, the “Gel point “is the term that usually used to describe the critical point, when the gel first forms²⁴ .

A PH electrode, Mettler Toledo Ultra-micro-ISM (the intelligent sensor management) read the PH of gelation, at which the titration was stopped (at PH threshold). HPLC grade water was added to complete the volume to 500µL, then the gels kept overnight in the fridge at temperature around 4℃ to equilibrate. The synthesis of hydrogels from these types of peptides is easy to prepare and affordable²² .

Titration curve:

The self-assembly of the peptides was determined through the PH range, thus the titration curve makes clear at which PH of the hydrogels formulated. NaOH at 0.5M was used for titration. A fixed volume has been added about 2 to 5µL, until the gels achieved. PH values recorded at each addition and the titration stopped at PH around 4.5-5, for all concentrations. In general, the titration stopped when a solid-like material was formed. Then the volume completed to 500μL with HPLC water. The samples are left in the fridge at 4℃, overnight to equilibrate and used on the following day.

Additionally, another titration has been done for the three peptides (P2, P3, P4) at concentration 30mg/mL, until reached PH from 9 to 10, to assess the volume of acidic media that required to make gel at basic PH higher than 5.The concentrations of peptides that have been titrated are 5, 10, 20, and 30mg/mL. The low solubility of the acidic form of the sodium salt is the main reason to use NaOH for titration. A 0.5M NaOH prepared by weighing 2gm of sodium hydroxide pellets (EMPLUR)®and then dissolved in 100mLof distilled water¹⁹. FTIR analysis was done for these concentrations²⁵.

Inverted vial test:

Hydrogel peptides have been tested at different concentrations (5,10, 20 and 30mg) each per 0.5mL of HPLC water, to investigate at which concentration the gel has been formed. The vial inversion test or gelation test was performed here to check the flowability of the hydrogels. After the hydrogels have been prepared in the microtubes are kept for equilibration at room temperature. Next, the microtubes sealed tightly and inverted for about 5 minutes, to test the gravitational flow²⁶ .

Fourier Transform Infrared spectroscopy (FTIR):

FTIR is a powerful technique used to study the functional group of the hydrogels. In other words, the structural information of the peptides was evaluated by this instrument²⁴. The secondary structure of the peptide hydrogels has been detected using a Thermo Nicolet IR 200 spectrophotometer. Different concentrations were analysed (5, 0, 20 and 30mg/0.5mL), about 15-20μL of the sample was loaded on the diamond stage. Regarding to analysis the transmission spectra, the OMNIC software was employed. The 4cmˉ¹ resolution, 128 scans for the sample background were performed and all were run at wavenumber from 400 to 4000cm. The effective spectra area was adjusted from 1450 to 1700cm.

Rheological Profile:

In terms of designing an injectable scaffold, the rheological study has a vital role, to test the recovery and stiffness. The oscillatory rheology for hydrogels can be measured using a Rheometer TA instrument, a stress-controlled Discovery Hybrid Rheometer HR-2. This device plays an integral part in the assessing the injectable delivery of hydrogels through establishing viscosity, storage, and loss values. To determine the mechanical features of peptide hydrogels, measuring the storage (G’) and loss (G’’) modulus as the amplitude and frequency sweep function. Storage modulus determines the stored energy through the process of material deformation. Thus, measure the stiffness and elasticity of the materials. While, loss modulus detects the properties of liquid material, the energy dissipated during the process²⁷. For this study, the frequency sweep from 0.01 to 15 HZ or 0.1 to 10 HZ, %strain at 0.2%, the temperature 25℃, and the gap fixed at 250μm. The samples are carried out in duplicates.

Calibration curve:

The doxorubicin as a drug model was used to prepare the calibration curve, 2mg (200μg) of doxorubicin dissolved in 10mL of HPLC water, to form the stock solution, that then utilised to prepare diluted concentrations (100, 50, 25, 12.5, and 3.062μg) completed to 2mL of HPLC water. The absorbance of these above concentrations was determined by using a Jenway 7315 UV/Vis spectrophotometer, the doxorubicin wavelength was fixed to 485nm, the experiment was carried out in triplicate.

In vitro studies of drug release:

Herein, to measure and control the doxorubicin release from the gel, 15mg of each peptide (P2, P3, and P4) was dissolved in 350μL of doxorubicin stock solution, and the volume adjusted to 500μL by HPLC water. A Jeway 7315 UV/Vis spectrometer was used to determine the absorbance. The wavelength range for doxorubicin is between 400 and 550nm, therefore the dependent spectra is at 485nm wavelength and1mL cuvette was used²⁸. The HPLC water was employed as the blank. The time intervals for drug release reading recording starting from the addition of buffer which is 1mL of HPLC water to the gel, then read the absorbance after 2, 6, 16, 24, and 48hour followed by converting the absorbance to the concentrations to plot the cumulative release curve using the equations of the calibration curve. The experiment carried out in duplicate, and for each sample the absorbance read in triplicate.

Scanning Electron Microscopy (SEM):

The morphological properties of the hydrogels such as the porosity and the nature of structure, can be analysed by SEM. The type of SEM that has been exploited to get images for the hydrogels and the Dox-loaded hydrogels is a JCM-6000 Plus Neoscope benchtop. SEM samples are generally need preparations before loaded, therefore, peptide hydrogels have been frozen at 4℃ for 1hour, followed by freeze drying for 2-3 days, using the Scanvac Cool Safe Freeze Dryer. After that the dried samples were fixed and coated by gold alloy, using the EMITECH K550X device for coating the hydrogels, then imaged in a SEM instrument.

RESULTS:

Preparation of hydrogels:

The hydrogels that have been prepared from the three types of the peptides are shown below, figure 2. The most significant features of these samples are the transparent nature, solid-like material, gel formation at specific PH range (within 4.5-5), and the variations in the flowability.

Figure 2: A photograph of hydrogels at different concentrations: 5, 10, 20, and 30mg, From left to right.

Titration curve:

Gelation of the peptides depends on the volume of the NaOH solution that has been added each time to the peptides, and PH recorded starting from 0 additions, until the formation of hydrogels, at which PH between 4.5-5. Hydrogels with PH-responsive can respond to different PH range within the gastrointestinal tract, therefore, they can also be used as oral drug delivery system. The average PH titration method indicates at which volume from the acidic media (NaOH), each peptide was required to form gel around PH 4.5-5, figure 3.

Figure 3: Titration curve of peptide conc.=30mg, illustrating the effect of volume added from 0.5M NaOH on the PH.

Inverted vial test:

Hydrogels have stimuli-responsive property with the ability to transformations. In other words, the volume and shape of the hydrogels are changed when subjected to swelling, shrinking and PH²⁹. The most notable part after preparation of hydrogels, is making inverted vial test (here used microtube), as evidence of forming gels. This test has been made for the four concentrations (5, 10, 20, and 30mg), for each peptide, figure 4.

Figure 4: gelation at different concentrations, 5, 10, 20, and 30mg/0.5mL from left to right. a: P2 hydrogels, b: P3 hydrogels, c: P4 hydrogels.

Fourier Transform Infrared spectroscopy (FTIR):

The peaks that have been appeared around 1629 cmˉ¹ is related to C=C stretching bond. The infrared region in the range from 3700 to 3000 cmˉ¹ is appeared for all peptides, and its attributed to O-H stretch bond of water³⁰ The focusing is on the spectra range from 1400 to 1800 cmˉ¹, figures 5, 6, 7, and 8. Additionally, the Y axis values were put in reverse ordered because these results are related to the inverted microtubes.

Figure 5: FTIR spectra for the P2 at the concentrations: 5, 10, 20, and 30mg. At PH: 4.30, 4.49, 4.52, and 4.90 respectively.

Figure 6: FTIR spectra for the P3 at the concentrations: 5, 10, 20, and 30mg. At PH: 4.78, 4.99, 5, and 5.4 respectively.

Figure 7: FTIR spectra for the P4 at the concentrations: 5, 10, 20, and 30mg. At PH 4.22, 4.61, 4.93, and 5.1 respectively.

Figure 8: FTIR spectra for the (P2, P3, and P4) + Dox, at 30mg and PH~5.

Rheological profile:

The mechanical properties of the peptide hydrogels have been detected using Rheometer-TA instrument, the results of the rheology are important, particularly, for the injectable hydrogels which act as a delivery vehicle to trigger the tumour. The samples that have been tested are peptide hydrogels at all concentrations and the hydrogels+ Dox. These samples loaded carefully onto the rheometer and the tests ran. The results from these tests are shown below, figures 9, 10, 11, and 12.

Figure 9: The rheological profile for the P2 concentration. 10, 20, and 30mg

Figure10: The shear modulus G’of P2, P3, and P4, at the same peptide hydrogel, at different concentrations

Figure 11: The shear modulus G’of P3, at different conc.

Figure 12: The shear modulus G’of P4, at different conc.

Drug release study:

The accumulated drug release was taken at different time points, to evaluate the anticancer activity at extended period³¹. The amount of Dox released from hydrogels was detected by UV-Visible spectrophotometry at 485nm, each hydrogel (peptide+ Dox) in the release kinetics study was conducted in triplicate³² .

The release profile of Dox is presented over 2, 6, 16, 24, and 48 hours, from the three types of peptides, fixing the same concentration for all peptides, to assist the effect of lysine residues on the cumulative drug release. It can also possible to consider that the diffusion, chemical, swelling and environment conditions might affect on the Dox release from the hydrogels. To understand the mechanism of drug (Dox) release from hydrogels, the release data was fitted to the Korsmeyer Peppas model. Figure 13..

Figure 13: The cumulative Dox release from the P2, P3 and P4 hydrogels.

SEM:

The images were taken in the range of micrometre length scales (50,100, and 200μm). The SEM showed the morphological, interior structures of the freeze-dried hydrogels. The SEM images revealed that water phase separation from the freeze-dried hydrogels and the pore size of the hydrogels are affected by the crosslinking, chemical composition and the drying conditions. SEM has been taken to the peptide hydrogels as well as peptides+ DOX, figure 14.

Figure 14: SEM for the P2, P3 and P4 hydrogels, from the left to right respectively + DOX.

DISCUSSION:

Photographs of the resulting hydrogels revealed that all the samples were transparent (no colour), however, this transparency tends to be nearly white as the concentration increased, hydrogels with lower concentration of peptides presented runoff. The most striking feature in the preparation particularly, the titration of the peptides, is the volume of the NaOH (0.5M), that has been added each time.

PH has a huge impact on the self-assembly of peptides, through affecting the ionization state and charges, via the electrostatic interactions. These peptides (P2, P3, P4) are PH response peptides, based on Zhang’s design, which form a 3-D Nano-fibrous network, and form gels of different stiffness, at above the critical gelation concentration^33,34.

It can possible to consider that PH also has effect on the storage modulus of the peptide hydrogel. Raising the PH to about 5, the storage modulus also increased ~1200 Pa, when the net charge was still lowered. In contrast, at PH 7, the values of storage modulus dropped to ~20 Pa, when non-charged peptide and the peptide starts to precipitate.

The peptide concentration plays an integral part in the mechanisms of the self-assembly peptides. The term of “Critical Self-assembly Concentration” or “Critical Aggregation Concentration” (CAC), has been used by scientists to describe the paramount importance of this factor. Peptides at CAC or above that will form nanostructures, while the dispersions formed at concentration lower than CAC.

Regarding to the results of the inverted test, it was obvious that the hydrogels or the viscous solutions were showed a transparent colourless nature, either in the bottom (for concentration ≥ 10mg) or in the top of the microtubes (concentration≤5mg). The most remarkable feature is the influence of the peptide concentration on the gel formation. To demonstrate, at 5mg concentration for all these three peptides were formed a weak gel, that on the inverted test and after 5 minutes the whole solution will flow and stay upside down the tube. In contrast, the gels have been formed at the three other concentrations: 10, 20, and 30mg, whereas the hydrogels were clear to observe, during the microtubes inversion the gels revealed no flow. However, the strength and texture of these gels were varied and gradually enhanced towards the highest concentration, which is the 30mg. This indicates that the CGC for these peptides is ≥ 10mg. The amphiphile peptides with the self-assembly property have been investigated using FTIR, to evaluate the presence of their secondary structure and the effect of PH and concentration on their functional groups. It is clear from the FTIR that the peptides revealed a significant peak at the region from around 1622 to 1635.80 cmˉ¹, these peaks are related to the C=O, C-N stretching and NH bending bonds, in the backbone of the peptides. Thus, this representing an evidence to the β-sheet structure of these peptides. These strong bands that appeared near 1630cm exhibit the antiparallel β-sheets.

Doxorubicin exhibited sp² hybridization of the carbon atoms. The FT-IR spectra for all peptides at different concentrations, showed peak at around 3400 cmˉ¹, it was related to the O-H bond, while the peak that appeared at 1620cmˉ¹ is related to the C=C or amide I bond.

A strong band at 1630cm and a weaker band near 1685cm are exhibited by the FTIR for the antiparallel β-sheet. According to the FTIR spectra, the position of the bands near 1622cm and 1635cm was not affected by the number of lysine residues, as observed the FTIR graphs showed the nearly same positions of these bands, at different peptides and concentrations that have been applied. However, it is remarkable that for higher concentrations 20mg and more clearly for 30mg, there were small band that positioned near 1580cmˉ¹. This is related to the lysine, NH3. The transmittance of the peptide hydrogels reflected the homogeneity of the structure and the interaction. In general, the imine group appeared at ~1568 cmˉ¹, and the bands around 3300 to 3500 cmˉ¹ were represented the stretching vibration of N-H and O-H. These peaks significantly increased as the number of linking in the hydrogels increased.

FTIR spectra of the hydrogels were indicated that the ordered β-sheet of the self-assembly peptides was increased with the concentrations, this is also emphases the importance of the CGC, which achieved at ≥10mg. However, there were slight variations among concentrations. This is because at low concentration (5mg) the single fibril was formed, and the connections among these fibrils were increased as the concentration increased. Thus, even at low concentrations (5 and 10mg), the bands of the β-sheet (1633-1685 cmˉ¹) have been appeared. It is clear from the FTIR spectra that the Dox loaded within the hydrogels has no effect on the β-sheet structure of the hydrogels, this is gives an advantage that Dox as a drug model has no impact on the functional group of the hydrogels.

Studies of drug release kinetics:

Doxorubicin occupied the free space that available among the network chain of hydrogels. Dox diffused through the hydrogel into the aqueous phase (HPLC grade water), during the drug release process. The diffusion and release of doxorubicin molecules followed the Higuchi equation. The EC50 of the Dox has been determined by researchers, to determine the amount of Dox loaded into the hydrogels. Determination of EC50 for different time points. From time point perspectives, the effect of Dox appeared within the first few hours, at higher concentrations. Doxorubicin after released and triggered the tumour cells, is supposed to interrupt the DNA synthesis and inhibit the mitosis. This is because Dox is an anthracycline anticancer agent that acts by intercalating into DNA. This is in turn causes apoptotic death of cancer cells. Controlled drug release from the hydrogel content can be achievable, via monitoring the PH in different sites of the body.

It is remarkable that from the results, the burst drug release has been achieved at the first few hours of Dox release into the added HPLC water (the buffer).

The exact release behaviour of Dox was dependent on the type of peptide (number of K residue), cation-π interactions and time point^34,35. To demonstrate, the highest Dox release from the hydrogels has been obtained from the P2 after 48hr, that is equal to 26.9%, this is because the P2 has the least number of lysine residues (only two), and thus it exhibited less cation-π interactions with the drug, when comparable to other peptides. In contrast, P4 at the same time point (48hr), was released about 17.55% of Dox, because this peptide has four lysine residues, which are as twice lysine number as P2, this means more interactions to hold the Dox among other peptides. The Dox was gradually released from P3, the average release=20.59% at PH~5 within 48hr, due to the breakage of cation-π bonds.

The SEM images displayed a fibre-like structure for the peptide hydrogels. Images of P2 hydrogels exhibited a less fibre structure than P3 and P4 hydrogels, the least homogeneous structure more like flakes. By contrast, P4 hydrogels showed the highest fibre-like structure (β-sheet) among peptides. However, all type of hydrogels exhibited similar network structures with minor differences in the pores size and linking number. The P4 hydrogels showed a smaller pore size, this is due to higher crosslinking (number of lysine) than other peptides.

CONCLUSION:

Hydrogels as vehicles showed a great promise for anticancer drug delivery. The gelation process is dependent on the peptide concentrations, time, and the external stimuli (PH). The major benefit of the PH and lysine-based hydrogels, is their ability to load the drug with poor aqueous solubility, then deliver and release it into the target tumour site, over a period of extended time. All these peptides did not form gel at concentration less than 10 mg/0.5mL.

The peptides with concentration of 5mg/0.5mL, there was no gel has been formed, even at high PH, they instead formed a viscous solution.

We can conclude that the peptides have ability to form gel, at PH from 4.5 to 5, and at certain concentration (above 5mg), which is called CGC, the effective peptide fibres have been investigated at that concentration.

Release profile of Dox can be greatly affected by varying the experimental and chemical parameters, such as the number of lysine residue (the type of peptides), time, concentration, and PH.

It is noticeable that Dox release from the hydrogel is PH dependent. SEM images were investigated the effect of crosslinker (number of lysine residues) on the morphological and topological properties of the hydrogels.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Singh, S., Dhyani, A. and Juyal, D. Hydrogel: Preparation, Characterization and Applications. The Pharma Innovation. 2017;6:pp.25-32.

2. Rani E., Ramadevi M., Usha A. An Overview on Hydrophilic three-Dimensional Networks: Hydrogels. Asian J. Pharm. Res. 2021; 11(1):23-28.

3. Bhushan P., Shashikant D., Mayur S. Citric Acid cross linked cellulose based Hydrogel for Drug Delivery. Asian J. Pharm. Res. 2017; 7(4): 247-255. doi.org / 10.5958/2231-5691.2017.00039.9

4. Buwalda SJ, Boere KW, Dijkstra PJ, et al. Hydrogels in a historical perspective: from simple networks to smart materials. Journal of Controlled Release : Official Journal of the Controlled Release Society. 2014 Sep;190:254-273. DOI: 10.1016/j.jconrel.2014.03.052. PMID: 24746623.

5. Zarzhitsky S, Rapaport H. The interactions between doxorubicin and amphiphilic and acidic β-sheet peptides towards drug delivery hydrogels. J Colloid Interface Sci. 2011 Aug 15;360(2):525-31. doi: 10.1016/j.jcis.2011.04.091. Epub 2011 Apr 29. PMID: 21575957.

6. Chen,J.and Zou,X. Self-assemble peptide biomaterials and their biomedical applications.Bioactive Materials;Bioactive Materials. 2019;4: 120-131. doi.org/10.1016/j.bioactmat.2019.01.002.

7. Devi N., Das M . Comparison Study of Blood Compatibility of an AMPS based Hydrogel with its Gold Nanoparticle Composite Hydrogel. Asian J. Research Chem. 2017; 10(6):750-756.doi.org/ 10.5958/0974-4150.2017.00127.4.

8. Roy A. Wahane A., Karankal S., Sharma P., Khutel D., Singh O., et al . Pharmaceutical Aspects on the Formulations of Hydrogel: An Update. Res. J. Pharma. Dosage Forms and Tech. 2018; 10(2): 79-84. doi: 10.5958/0975-4377.2018.00012.5.

9. Kopecek J. Hydrogel biomaterials: a smart future? Biomaterials. 2007 Dec;28(34):5185-92. doi: 10.1016/j.biomaterials.2007.07.044. Epub 2007 Aug 13. PMID: 17697712; PMCID: PMC2212614.

10. Ahmed EM. Hydrogel: Preparation, characterization, and applications: A review. J Adv Res. 2015 Mar;6(2):105-21. doi: 10.1016/j.jare.2013.07.006. Epub 2013 Jul 18. PMID: 25750745; PMCID: PMC4348459.

11. Sneha V. Sawant, Shirish V. Sankpal, Kisan R. Jadhav, Vilasrao J. Kadam. Hydrogel as drug delivery system. Research J. Pharm. and Tech. 5(5): May2012; Page 561-569.

12. Mastria EM, Cai LY, Kan MJ, Li X, Schaal JL, Fiering S, Gunn MD, Dewhirst MW, Nair SK, Chilkoti A. Nanoparticle formulation improves doxorubicin efficacy by enhancing host antitumor immunity. J Control Release. 2018 Jan 10;269:364-373. doi: 10.1016/j.jconrel.2017.11.021. Epub 2017 Nov 13. PMID: 29146246; PMCID: PMC6475912.

13. Hajare AA, Powar TA. , Bhatia NM., More HN. Development and Validation of RP-HPLC Method for Determination of Doxorubicin Hydrochloride from Vacuum Foam Dried Formulation. Research J. Pharm. and Tech. 2016; 9(9):1352-1356.

14. Ahmed, A. Z., Shetty, P., Satyam, S. M., D’souza, M. R., Herle, A. M., and Singh, V. K. Methyl gallate mitigates doxorubicin-induced peripheral cytopenias: A preclinical experimental study. Research Journal of Pharmacy and Technology. 2021;14(9), 4529-4534. https://doi.org/10.52711/0974-360X.2021.

15. Saito, H., Hoffman, A.S. and Ogawa, H.I. Delivery of Doxorubicin from Biodegradable PEG Hydrogels Having Schiff Base Linkagest. Journal of Bioactive and Compatible Polymers. 2007; 22(6):589-601.

16. Seib, F.P., Pritchard, E.M. and Kaplan, D.L. Self-Assembling Doxorubicin Silk Hydrogels for the Focal Treatment of Primary Breast Cancer. Advanced Functional Materials. 2013;23(1):58-65.

17. Dadsetan, M.,Liu,Z.,Pumberger, M.,Giraldo, C.V.,Ruesink, T.,Lu, L.andYaszemski, M.J. A stimuli-responsive hydrogel for doxorubicin delivery. Biomaterials. 2010; 31(31): 8051-8062.

18. Chen, Z., Zhang, P., Cheetham, A.G.,Moon, J.H., Moxley, J.W., Lin, Y. and Cui, H. Controlled release of free doxorubicin from peptide-drug conjugates by drug loading. Journal of Controlled Release. 2014;191:123-130.

19. Ratnaparkhi M.P., Andhale R.S., Karnawat G.R. Nanofibers – A Newer Technology. Research Journal of Pharmacy and Technology. 2021; 14(4):2321-7.

20. Matson, J.B., Newcomb, C.J., Bitton, R. and Stupp, S.I. Nanostructure-templated control of drug release from peptide amphiphile nanofiber gels. Soft Matter. 2012; 8 (13): 3586-3595.

21. Mangione, M.R.,Giacomazza, D., Cavallaro, G., Bulone, D., Martorana, V.and San Biagio, P.L. Relation between structural and release properties in a polysaccharide gel system. Biophysical Chemistry. 2007;129(1):18-22.

22. Nandi N, Gayen K, Ghosh S, Bhunia D, Kirkham S, Sen SK, Ghosh S, Hamley IW, Banerjee A. Amphiphilic Peptide-Based Supramolecular, Noncytotoxic, Stimuli-Responsive Hydrogels with Antibacterial Activity. Biomacromolecules. 2017 Nov 13;18(11):3621-3629. doi: 10.1021/acs.biomac.7b01006. Epub 2017 Oct 30. PMID: 28953367.

23. Ligorio, C., Zhou, M., Wychowaniec, J.K., Zhu,X.,Bartlam, C., Miller, A.F., Vijayaraghavan, A., Hoyland, J.A. andSaiani, A. Graphene oxide containing self-assembling peptide hybrid hydrogels as a potential 3D injectable cell delivery platform for intervertebral disc repair applications. Acta Biomaterialia. 2019;92:92-103.

24. Enev, V., Sedlacek, P., Jarabkova, S.,Velcer, T. and Pekar, M. ATR-FTIR spectroscopy and thermogravimetry characterization of water in polyelectrolyte-surfactant hydrogels. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2019; 575.

25. Rana R., Kumar A., Bhatia R. Impact of Infra Red Spectroscopy in Quantitative Estimation: An Update. Asian J. Pharm. Ana. 2020; 10(4):218-230. doi: 10.5958/2231-5675.2020.00040.X

26. Yeh, M., Zhao, J., Hsieh, Y., Lin, J., Chen, F., Chakravarthy, R.D., Chung, P., Lin, H. and Hung, S. Reverse thermo-responsive hydrogels prepared from Pluronic F127 and gelatin composite materials. RSC Advances. 2017;7(34): 21252-21257.

27. Pont, G., Chen, L., Spiller, D.J.The effect of polymer additives on the rheological properties of dipeptide hydrogelators. Soft Matter. 2012; 8:7797-7802.

28. C A Schoener, B Carillo-Conde, H N Hutson, N A Peppas. An inulin and doxorubicin conjugate for improving cancer therapy. J Drug Deliv Sci Technol. 2013;23(2):111-118.

29. Kharlampieva, E., Kozlovskaya, V., Zavgorodnya, O., Lilly, G.D., Kotov, N.A. and Tsukruk, V.V. pH-responsive photoluminescent LbL hydrogels with confined quantum dots. Soft Matter. 2010; 6(4): 800-807.

30. Thurmer, M.B., Diehl, C.E., Brum, F. and Santos, L.A.D. Preparation and characterization of hydrogels with potential for use as biomaterials.Materials Research. 2014;17:109-113.

31. Jiang, B., Larson, J.C., Drapala, P.W., Perez-Luna, V.H., Kang-Mieler, J.J. andBrey, E.M. Investigation of Lysine acrylate containing poly(N-isopropylacrylamide) hydrogels as wound dressings in normal and infected wounds. Journal of Biomedical Materials Research Part B: Applied Biomaterials. 2012; 100(3): 668-676.

32. Zhang, Y., Yang, C., Wang, W., Liu,J., Liu, Q., Huang, F., Chu, L., Gao, H., Li, C., Kong, D., Liu,Q.and Liu, J. Co-delivery of doxorubicin and curcumin by pH-sensitive prodrug nanoparticle for combination therapy of cancer. Scientific Reports. 2016; 6(1).

33. Zhang,Y.N., Avery, R.K., Vallmajo-Martin, Q., Assmann, A., Vegh, A., Memic, A., Olsen, B.D., Dr.Annabi, N., Khademhosseini, A., A Highly Elastic and Rapidly Cross-linkable Elastin-Like Polypeptide-Based Hydrogel for Biomedical Applications. Advanced Functional Materials. 2015;25: 4814-4826.

34. Mishra A., Sahu G., Kumar A. , Patel D., Rathore G., Sahu D., Diwan M., Kanwar D. Underlining the pharmaceutical aspects associated with the development of pH responsive hydrogel. Research J. Pharm. and Tech. 2017; 10(4): 1261-1268.

35. Watkins, K.A. and Chen, R. pH-responsive, Lysine-based hydrogels for the oral delivery of a wide size range of molecules. Int J Pharm. 2015; 478(2): 496-503.

Received on 11.06.2022 Modified on 21.10.2022

Research J. Pharm. and Tech 2023; 16(4):1797-1805.

DOI: 10.52711/0974-360X.2023.00295