DOI: 10.31038/JPPR.2021435

Abstract

The development of bioactive hydrogels has received much attention in the field of tissue regeneration. In the study, we utilized an injectable and biocompatible chitosan-Poloxamer P407 (CS-P407) hydrogel to deliver dual amino acids: glutamic and arginine. The amphiphilic CS-P407 copolymer structure was identified by ¹H-NMR and FITR. The obtained copolymer solution shows the sol-gel transition point at body temperature (35-37°C), which is suitable for wound healing application. Through SEM imaging, this hydrogel presented a well-defined three-dimensional microporous network. In addition, CS-P407 exposed excellent bio-compatibility, with 90% fibroblast cell survival. The encapsulation of Arg and/or Glu did not induce any change to sol-gel transition behavior of CS-P407 as well as their biodegradation. The release of Arg and Glu from CS-P407 performed a sustainable profile following the non-Fickian kinetic model. The bioactive hydrogel may provide great potential for future clinical chronic wound management.

Keywords

Arginine, Chitosan, Glutamic, Hydrogel, Poloxamer P407, Wound healing

Introduction

Currently, the engineered bio-materials have significantly contributed to the field of tissue regeneration [1]. Specifically, engineered biomaterials, can control, regulate or mimic the natural regeneration process of tissue or organ. During this process, engineered biomaterials provide the framework structure to promote the migration and the proliferation of the target cells resulting in the promotion of the re-programming tissue [1,2]. In terms of mimicking extracellular matrix, the hydrogel is known as the best candidate in tissue engineering [2,3]. Hydrogels have a 3D network structure of hydrophilic polymeric with a controllable mechanical property as well as the ability to release growth factors sustainably; consequently, supporting the healing of damaged tissues [2-4]. More recently, hydrogel response to changing temperature through physical cross-linking has attracted extensive studies [5]. The outstanding advantages of the temperature-responsive hydrogel are of relative ease and do not require exogenous agents that may induce immune responses inside the body [5]. Additionally, the sol-gel transition behavior of thermal-sensitive hydrogel provides an injectable platform with minimal invasion compares to surgical delivery [4,5].

In the modern concept of tissue engineering, hydrogel-based natural materials such as chitosan, alginate, gelatin, or collagen have been fabricated [1-3]. Among them, chitosan is of great significance for tissue regeneration [5]. Chitosan has good biocompatibility, low toxicity, and rapid biodegradability. Various studies proposed the great pharmaceutical application of chitosan, including anti-bacteria, anti-inflammation, hemostasis, etc. [6,7]. Furthermore, chitosan contains abundant functional groups on its backbone; therefore, it is easy to modify or fabricate to optimize the hydrogel structure [6-8]. For example, chitosan could be co-polymerization with pluronic F127 to form the thermal sensitive hydrogel [6,8]. The system was considered an effective platform for drug delivery and tissue regeneration [5-7]. Poloxamer, an amphiphilic, thermo-sensitive, and FDA-approved Triblock copolymer of poly (ethylene oxide) and poly (propylene oxide), is one of the most studied platforms for the preparation of highly efficient hydrogels [5,6,8]. Chitosan-grafted poloxamer is widely exploited in cartilage regeneration, wound healing, burn healing, and anti-cancer drug delivery [7,9-13].

L-Arginine (Arg) is an essential amino acid that helps to prevent and treat circulatory diseases, alleviate fatigue, and stimulate the immune system. Arg is also known as endothelial nitric oxide synthase enzyme (eNOS) substrate [14], responsible for Nitric Oxide (NO) synthesis [15]. NO creates the signal for macrophage activation leading to the migration of fibroblast cells in wound healing process [14]. Along with Arg, L-Glutamic acid (Glu) is a necessary amino acid in the body, a precursor for collagen synthesis [15]. Collagen is a crucial protein for skin and tissue regeneration. It has been shown that the rate of collagen synthesis in tissues was completely dependent on the concentration of Arg and Glu in the micro-environment [16,17]. Therefore, Arg and Glu supplements into the hydrogel are necessary to ensure treatment outcomes of wounded areas. In this study, we develop a thermo-sensitive system of chitosan-poloxamer (CS-P407) hydrogel with Arg and Glu dual loading. The thermal-responsive behavior of the multifunctional hydrogel was evaluated by inverted tube method. The release profile with the kinetic model of Arg and Glu was also exanimated. Furthermore, the degradation of this system was investigated in the physiological medium. It is expected that CS-P407 in the combination with two amino acids could be used as a promising functional wound dressing in the future clinical treatment of chronic wounds.

Materials and Methods

Chemicals

Chitosan (CS) low molecular weight 85% deacetylated was supplied from Sigma. L-Arginine (Arg), L-Glutamic (Glu) and FMOC chloride (FMOC-Cl) were purchased from Sigma-Aldrich (USA). The cellulose dialysis membranes (molecular weight cut-off of 12-14 and 3.5 kDa) obtaining from Repligen were used to purify products. Mononitrophenyl formate-activated Poloxamer (NPC-P407-OH) was prepared in our previous studies at the Institute for Applied Materials Science as described [12,13]. All other chemicals were purchased from Thermo Fisher Scientific (Waltham, MA) or Fisher (USA) or of the analytical grade.

Synthesis of Poloxamer P407-Conjugated Chitosan Copolymers (CS-P407)

CS-P407 was prepared by the combination of Poloxamer P407 activated by mononitrophenyl formate with CS solution in acidic media (pH 4-5) followed by our previous report [9]. Briefly, NPC-P407-OH 10°C was added dropwise into CS solution (mass ratio of CS: NPC-P407-OH was 1:15). After 24 h, CS-P407 was dialyzed against distilled water using a cellulose membrane (MWCO = 12-14 kDa) in 5 days and then lyophilized to obtain the final product. ¹H-NMR and Fourier Transform Infrared Spectroscopy (FTIR) measurements were used to identify the chemical structure of copolymers.

Characterizations of CS-P407 Hydrogels

Preparation of the CS-P407 Hydrogels and Bioactive Hydrogels

The grafted copolymer CS-P407 was dissolved in DI water (PBS, DMEM) contained vials (5, 8, 10, 12, 15, and 20% w/v) at 4°C. The temperature-responsive behavior of copolymer was investigated by sol-gel transition observation with a temperature range from 15 to 50°C, each measurement is spaced 5°C. The temperature of the incubator was stabilized in 5 min before dipping the test tube. At each investigated temperature, the test tube was kept in the incubator for 10 min to observe the gel-sol transition behavior [16,17]. The sol-gel transition of the bioactive hydrogels was conducted in the same method. The three-dimensional microporous network of the hydrogel was observed by SEM.

Adhesion Testing of the CS-P407 Hydrogel

The pigskin was cleaned of fat and cut into 2 squares with dimensions of 2.5 x 3 cm then soaked in 1X PBS solution (pH 7.4) for 2 h at 37°C. The pigskin was then fixed on the glass slide (25.4 x 76.2 cm) and left to stabilize for about 1 hour. The hydrogel (0.5 g) was drenched in cold water to evenly coat the pork skin. The remaining pigskin is then placed on top of the hydrogel-coated skin. The sample was stabilized at 37°C then pulled with a universal tester (Portable Tension Tester MTT 1500) at 10 mm/min until the piece of skin is separated. The value of the adhesive strength is calculated as the tensile strength at the point of the separated skin divided by the contact area of the skin. The unit of adhesive strength is measured in N/mm², then converted to KPa (1 N/mm² = 1000 KPa). The experiment was repeated thrice.

Cytocompatibility Test of the Hydrogels

Human fibroblasts cells (BJ (ATCC® CRL-2522™) were used in this study. The percentage of viable cells was determined by Sulforhodamine B (SRB) assay. The process was based on the guidance of Abcam. CS-P407 hydrogel was dissolved in distilled water then irradiated with a dose of 25 kJ for sterilization. The copolymer solution (100 µL) was evenly seeded in the culture disks with the dense of 10⁴ cells/mL, then the culture media DMEM with the supplement of 10% FBS and 1% Penicillin Streptomycin was added to reach 0.5 mL. The untreated cells incubating with completed DMEM only was used as the control while the cell culture with 10 µL water was considered as the negative control and the cell culture with 10 µL Camptothecin (CPT) solution at the determined concentration (0.2 µg/mL) was considered as the positive control. The cells were incubated at 37°C, 90% humidity, 5% CO₂ condition. At the designed time (4h, 24h, 72h, and 96h), the SRB kit was applied to each well. The results were recorded by ELISA at 570 nm. The percentage of viable cells was determined by the ratio of OD value of the treated cells to untreated cells. All the experiments were repeated thrice each case with 3 replications.

Release Profile and Release Kinetics of Amino Acid

CS-P407 Hydrogel Containing Amino Acid

The fabrication of Glu/Arg loaded CS-P407 hydrogel. CS-P407 (4.5 g) was dissolved in 20.5 mL DI containing Glu/Arg at 10°C. Copolymer solution with predetermined Glu/Arg concentration was lyophilized for further use in Glu/Arg content evaluation and in vitro release behavior. Since it is difficult to determine the content of Glu/Arg loaded CS-P407 hydrogels, a mediated-reagent (FMOC-Cl) was used to quantify different amino acids via reactions between amino acids and FMOC-Cl [18-20]. Pure Glu/Arg was dissolved in H₂O with various concentrations (in the range from 1 to 10 ppm). The other stock solutions were prepared by dissolving 1.237 g H₃BO₃ in 100 mL H₂O with pH was adjusted to 9 by NaOH 0.1 M and 100 ppm FMOC-Cl in acetonitrile. A mixture containing 3 stock solutions as follows Glu/Arg: H₃BO₃ : FMOC-Cl = 1:1:2 (v/v) was stirred at 30°C in 2h. The residual FMOC-Cl and FMOC-OH were eliminated by diethyl ether (5×5 mL). The remaining solution was diluted to 10 mL. UV-Vis spectra (Agilent 8453 UV-Vis Spectrophotometer) were used to determine each amino acid content at the absorb wavelength of 265 nm. The release profiles of loading agents from the hydrogels in vitro were characterized by self-diffusive method of Glu/Arg-loaded hydrogel contained in a cellulose membrane (MCWO = 3.5 kDa). Samples (2 mL hydrogel 18%) was immersed in the phosphate-buffered saline (PBS) pH 7.4 (20 mL) and shaken (100 rpm) at 37 ± 1°C. At predetermined intervals (0h, 1h, 3h, 5h, 7h, 9h, 12h, 24h, 36h, 48h), the released PBS was collected and replaced by fresh PBS. The number of released agents was measured using UV-vis spectrophotometer as mentioned. The experiments were repeated 3 times. The percentage of released Glu/Arg was calculated as follows:

Where Cn is the concentration of Glu/Arg in the sample, Cn-1 is the concentration of Glu/Arg released at time t, Vs is the volume of incubation medium, and Vt is the volume of medium replaced at time t [21].

The release profiles of Glu/Arg were found to be suitable for zero and first-degree equations, Higuchi, and Korsmeyer [22,23]. The mean dissolving time (MDT) value was calculated from release kinetic data using equation (Mockel and Lippold) [24].

Where n and k are the release exponent and the release rate constant from the Korsmeyer equation, respectively.

Degradation Profiles

Approximately 2 mL hydrogels (Mi) were fabricated in vials subsequently incubated at 37°C. The samples were prepared in distilled water and then diluted in 5 mL buffer PBS pH 7.4 or DMEM. After every 2 days incubation, the liquid was consecutively replaced by 5 mL culture media until the hydrogels had completely disintegrated. The weight of the remaining hydrogel is recorded by an electronic balance. The data profiles were expressed as the average of three measurements. The percentage weight loss is calculated as follows:

M_i: initial gel mass (g); M_t: the remaining gel mass (g) and after the degraded time.

Results and Discussions

Characterizations of the Amphiphilic CS-P407 Copolymers

CS-P407 is synthesized based on the carbamate group synthesis reaction through a covalent bond between the carbonate group (NPC-P407-OH) and amino group (-NH₂) of CS (Figure 1). The ¹H-NMR spectroscopy of CS-P407 copolymer has been mentioned in our previous research [25,26]. Overall, the resonance signal of Poloxamer protons and methyl, methylene and methine group exist in 3.41 – 4.03 ppm and 1.10 ppm (-CH₃ of PPO unit). The max resonance was achieved at δ = 1.97 ppm, δ = 2.90 ppm, and δ = 4.64 ppm for protons in glucosamine of CS backbone, which confirmed the structure of co-conjugated compound CS-P407. The FTIR result (Figure 2) describes the spectroscopic features of standard CS in 3368.02 cm^-1 wavenumber due to oscillation of O-H bond, 1558.89cm^-1 is the oscillation of N-H bond. The NPC-P407-OH spectroscopy shows that the peak in 2885.96cm^-1 belongs to the C-H bond in the PEO fraction of Poloxamer P407. The peak at 1111.03cm^-1 is caused by a specific C-O linkage of Poloxamer P407. These signals also occurred in CS-P407 spectroscopy at 2890.07cm^-1 is a peak of C-H linkage. The stretched oscillation of the C-O bond shows a signal at 1111.88cm^-1. The peak at 1571.05cm^-1 indicates the -NH deforming signal of the amine group in CS, even though this signal doesn’t exist in CS-P407 spectroscopy due to the amine group has formed a bond with P407 and create an amide I group with a wavenumber at around 1650.04cm^-1 [27]. To sum up, the FTIR spectroscopy data shows that the peaks observed are suitable with the expected functional groups in the compound structure of conjugated copolymer.

Figure 1: Synthesis of thermosensitive copolymer CS-P407

Figure 2: The FTIR spectra of P407, NPC-P407-OH, and CS-P407.

Characterizations of CS-P407 Hydrogels

The CS-P407 hydrogel was coated with a thin layer of approximately 2 mm on the glass slide and the samples were allowed to dry naturally. The sample was then measured by SEM to observe the hydrogel surface structure.

SEM imaging results of Figure 3 show that the CS-P407 hydrogel has an abundant-porous structure (1-2µm) formed by the overlapping network of CS-P407 copolymers. This is the most important characteristic of the hydrogel system (scaffold) since it is not only able to absorb the fluid exudate and maintain certain water content in the wound but also allows the fibroblasts to divide and migrate. The interconnected porous structure has an impact on the supply of nutrient and gas exchange in order to maintain cellular ingrowth and retain a high amount of water, and also it offers ideal material for the Glu/Arg delivery carrier system [10].

Figure 3: C-P407 hydrogel surface structure (x1000 image on the left) and (x5000 image on the right)

Thermal Sensitivity of CS-P407 Solution

Obtained results of sol-gel transition investigation show that CS-P407 solution can create gel at a relatively low copolymer concentration (above 10% wt/v) at 32-37°C (Figure 4A). When replacing water with PBS buffer and DMEM cell culture media, the transition temperature is unaffected significantly. The thermosensitive hydrogel was formed via hydrophobic interaction of hydrophobic PPO domains in the CS-P407 as seen in Figure 4B. The phenomenon was reported in several studies [5,7,9,10].

Figure 4: The phase diagram of the sol-gel transition (A); illustration of the sol-gel transition of the CS-P407 solution (B).

Adhesion of the Hydrogel Scaffolds

The cohesive ability of hydrogel material to the skin surface is a critical factor in creating material for wound healing. The cohesion process will suppress plasma leakage, prevent bacterial infection, maintain gaseous exchange and provide better access to bioactive compounds in the hydrogel. The tissue adhesive experiment was conducted in porcine skin. As shown in Figure 5, the adhesivity of CS-P407 hydrogels at 15%Wt, 18%Wt, and 20%Wt are 5.51 ± 0.88 KPa, 6.62 ± 0.7 KPa and 5.74 ± 0.74 KPa, respectively. This result was similar to Ji Hyun Ryu’s research in 2011 (The value in this study are about 5.3±2.6 KPa) [28]. At the same condition, P407 hydrogel exhibits a very low tissue adhesion, at which the value is 0.79 ± 0.26 KPa that is similar to a previous study (0.72±0.32 KPa) [29].

Figure 5: Adhesion strength (KPa) of the material to the pigskin surface.

The experiment was repeated three times independently (n=3), and the errors are presented in S.E. Statistical significance of p < 0.001 is indicated by ***. Non-statistical insignificance of p ≥ 0.05 is indicated by ns.

This adhesive feature is formed by the positively charged amine groups in the CS backbone interact with the collagen matrix of porcine skin. It depends on chitosan, a polymer with strong adhesivity through its -NH₂ groups. As a result, when attaching to the skin surface, CS-P407 can interact with the surface through hydrogen bonds, electrostatic bonds, hydrophobic interaction that make CS-P407 have better adhesive intensity.

Cytotoxicity of Hydrogel CS-P407

In biomedical material, biocompatibility, or the fitness and harmlessness of the material with the human body and related physiological activities, is the primary criteria to decide its possibility for application. Fibroblasts are vital for wound regeneration, especially in the division, differentiation, and migration stage of fibrocytes at the wound surface. These cells synthesize and secrete extracellular proteins, mainly collagen, to reconstruct the extracellular matrix of connective tissue. In this research, a preliminary cytotoxicity assessment was conducted on fibroblast cell lines from human skin. According to the results in Figure 6, CS-P407 hydrogel is non-toxic for fibroblast. CPT was used at 3 µg/mL, the fibroblast growing rate decreased 42.02 ± 8.05% after 4h of exposure and go down to 7.21 ± 5.38% after 24 hours with total cell death was observed after 48 hours. The number of cells on CS-P407 increase from 106.07 ± 3.52% after 4 hours to 136.8 ± 5.07% after 48 hours. This proves that besides its non-toxicity, CS-P407 can improve cell growth for fibroblasts.

Figure 6: Percentage of BJ fibroblasts growth incubated in 0.1 mL of CS-P407, water (negative control) and CPT (positive controls).

In Vitro Amino Acid Release Profile and Release Kinetic Models

In this study, we investigated the proliferative capacity of human fibroblasts (ATCC® CRL-2522™) of free Arg and Glu. Tested concentrations of Arg and Glu from 0-250 µM and 0-500 µM respectively. Figure S.1 showed that the increasing concentration of active ingredients leads to cell proliferation, proving that both Arg and Glu support cell growth and increase wound healing. Arg’s support was significantly impactful on proliferation. Specially, 50 µM for Arg (number of cells = 9 x 10⁴) and 250 µM for Glu (number of cells = 8 x 10⁴) are the optimal concentration for cell proliferation. On the other hand, the excessive use of Arg and Glu also reduces cell growth. This inhibition of cell growth can be seen above 200 µM for Arg and above 450 µM. Based on obtained data, we selected the optimal carry concentrations of Arg and Glu are 50 µM and 250 µM, respectively, into CS-P407. These primary results are the premise to develop a hydrogel system CS-P407 to support treatment and wound healing, show great applications in medicine in general and tissue regeneration in particular.

The gelation temperature range was determined from the minimum temperature of gel formation T_gel to the temperature at which the gel begins to melt T_m. In Figure 7, from the measured results, it can be seen that the T_gel gelation temperature of CS-P407 carrying amino acids when mixed with distilled water is higher than that of PBS night medium and physiological DMEM medium at the same concentration. Gel forming temperature range in an aqueous medium is mostly narrower, the results are similar to that of CS-P407 hydrogel. The influence of the medium on the gelation of the hydrogel can be seen. It is understood that in the medium PBS and DMEM gel has higher mechanical properties, more stable. The above results show that the gel is effective in carrying amino acids, without losing the inherent mechanical properties of the system. The CS-P407 polymer system creates a gel at 30-35°C and the gel melting point is above 50°C, so it is very suitable for biomedical applications, especially wound treatment gels.

Figure 7: The phase diagram of the sol-gel transition of CS-P407/Arg (A); CS-P407/Glu (B) và CS-P407/Glu/Arg (C)

Figure 8 represents the release rate of free Glu and Arg after 12 hours achieve 100% when CS-P407/Glu, CS-P407/Arg, and CS-P407/Glu/Arg rates are 32.53%, 47.28%, and 65.02%. After 48 hours, the release rate at CS-P407/Glu and CS-P407/Arg reach 43.97% and 96.83%, respectively. The Arg release profile of CS-P407 was faster compared to Glu. This difference is according to the carboxylated form of the 2 -COOH group in Glu at pH=7.4 has electrostatic interaction with the positively charged structure of CS-P407, which leads to a slower release rate. When both amino acids were captured in the hydrogel, their release rate reaches 80.59% after 48 hours. Thus, CS-P407 hydrogel helps decelerate the release rate of amino acids, increases their absorptivity at wounded tissue

Figure 8: Release profile of amino acid (Glu/Arg) from the hydrogel CS-P407

Among all formulas investigated, the Korsmeyer-Peppas regression model has the greatest fitness (R²= 0.9155-9.8874) (Table 1 and Figure 9). With this model, the transport exponent (n) belongs to the (0.3078-0.5218) interval, indicating that the release mechanism of free amino acid and CS-P407/Glu are Fickian diffusions. With free amino acid, it is the passive diffusion, for CS-P407/Glu is due to the CS-P407 gel barrier interaction with Glu. On the other hand, with CS-P407/Arg and CS-P407/Glu/Arg, the release mechanism is non-Fickian, influenced by diffusion and swelling. The rate of diffusion and swelling are the same. The rearrangement of polymer chains happens in slow progress, while the diffusion triggers some abnormal effects over time [30,31].

Table 1: Amino acid release parameters of CS-P407/Glu, CS-P407/Arg, and CS-P407/Glu/Arg hydrogel were obtained to four different mathematical models of drug release kinetics.

Formulation	Mathematical models for drug-release kinetics
	Zero order		First order		Higuchi		Power law
	k0	R2	k1	R2	kH	R2	K	n	R2
Glu Free	0.0981	0.9836	0.5243	0.9459	0.2911	0.9980	0.3179	0.4122	0.9972
Arg Free	0.0935	0.9972	0.5182	0.9854	0.2771	0.9846	0.3339	0.3760	0.9760
CS-P407/Glu	0.0121	0.9231	0.1560	0.8463	0.0799	0.9790	0.1364	0.3078	0.9920
CS-P407/Arg	0.0238	0.9875	0.1805	0.9188	0.1369	0.9982	0.1422	0.4719	0.9974
CS-P407/Glu/Arg	0.0233	0.8275	0.1869	0.7269	0.1611	0.9195	0.2032	0.5218	0.9155

Figure 9: Release kinetics of Glu/Arg from CS-P407 fitted to four kinetic models: (A) zero-order kinetic model, (B) first-order kinetic model, (C) Higuchi model, and (D) Korsmeyer-Peppas model.

Table 2 shows that the MDT value of hydrogel is much higher than free amino acid, which means that amino acids have been entrapped inside the hydrogel matrix. This improves the usage efficiency of the bioactive compound with a short half-life. Thus, CS-P407 hydrogel has prominent potential in the drug distribution process in wound treatment.

Table 2: The fit kinetic model and the MDT value of the modulation formulas.

Formulation	Order of release	t25% (hours)	t50% (hours)	t75% (hours)	t90% (hours)	MDT (hours)
Glu Free	Fickian	0.5583	3.0000	8.0223	12.4849	4.7056
Arg Free	Fickian	0.4631	2.9265	8.6043	13.9741	5.0533
CS-P407/Glu	Fickian	7.1578	68.0652	254.1572	459.6082	152.3172
CS-P407/Arg	Non-Fickian	3.3073	14.3690	33.9318	49.9357	20.0140
CS-P407/Glu/Arg	Non-Fickian	1.4878	5.6165	12.2164	17.3257	7.2699

Degradation Profiles

The hydrogel degradability was evaluated by gravimetric analysis of CS-P407 in PBS (pH 7.4) or DMEM solution until the hydrogel was completely degraded. Figure 10 shows that CS-P407 hydrogel had a degradation time of 10 days in PBS buffer with pH 7.4, which was 1.7 times longer than the P407 hydrogel sample. This shows that the combination of Poloxamer P407 with CS (which is a bio-adhesive agent) has increased the stability of the hydrogel system. The results can be explained as the interweaving of the branched copolymer chains is increased by CS, leading to increase hydrogel stability during decomposition. Comparing degradation time in two physiological media, PBS medium pH 7.4 degraded more rapidly, when all samples have been downgraded after 12 days. DMEM medium has a longer degradation time, as the hydrogel sample remains after 16 days of investigation. Loading arginine and glutamic resulted in prolonging degradation of the bioactive hydrogels. This could be contributed by hydrogen bonding formation of amino acids and chitosan chains leading to increase the stability of the hydrogel matrix.

Figure 10: Degradation profile of hydrogel samples in PBS pH 7.4(A) and DMEM (B).

Conclusion

The bioactive hydrogel loading glutamic and arginine amino acid was developed. CS-P407 18 wt/wt% copolymer solution strongly forms hydrogel at body temperature. The system exhibits a porous structure, high tissue adhesion, and cytocompatibility which is injectable for applying minimal invasion surgery. The optimum concentrations of Arg and Glu used in the CS-P407 system to increase cell proliferation are 50 µM and 250 µM, respectively. The CS-P407 hydrogel performed a suitable platform for controlling the delivery of these amino acids. The release behavior is affected by concentration diffusion and the swelling of the gel system. Arginine and glutamic encapsulation resulted in the prolonged degradation of bioactive hydrogels. The preliminary results could pave the way to apply the bioactive hydrogel in would healing.

Acknowledgements

This work was supported by the Ho Chi Minh Department of Science and Technology (number of contract 47/2019/HĐ-QPTKHCN)

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DOI: 10.31038/JPPR.2021434

Abstract

A novel non-invasive approach for treatment of localized obesity is introduced utilizing carbopol gel containing soybean phosphatidylcholine based nanovesicular system for topical application. The tested systems are designed to combine the absence of side effects of the multi-injection system used in mesotherapy and the ease of application. Nanovesicles such as transfersomes and transethosomes were prepared using soybean phosphatidylcholine, tween 80, sodium deoxycholate, cremophor, and oleic acid in different concentrations determined according to 3D-optimal mixture experimental design. The prepared vesicles were evaluated and incorporated into carbopol gel. The stability of the prepared nanovesicles and gel was examined after storage for six months at 4˚C. In-vivo and In-vitro studies were performed using male albino rats. Performed experiments on rats showed that the three formulations of choice (F4e, F11e and Et11) succeeded to reduce body weight, percentage dorsal fat and total lipid content significantly (P<0.05) without appearance of any sign of skin irritation compared to PC marketed injections (Adipoforte^®) used in mesotherapy. PC nanovesicular gel, containing transethosomes (F4e, F11e & Et11), can be significantly considered as effective noninvasive treatment for localized obesity as an alternative to multi-injections for mesotherapy.

Keywords

Phosphatidylcholine, HPLC-Determination, Experimental Design, Nanovesicles, Mesotherapy.

Introduction

Obesity is recognized as one of the most important public health problems facing the world^[1]. Up to 30% of the western adults are obese^[1]. In Middle East and North Africa, obesity prevalence reaches about 19% of whole population while it reaches 33% in Egypt [2]. Mesotherapy is a controversial cosmetic procedure for localized fat accumulations reduction. Subcutaneous Phosphatidylcholine(PC) injection has been performed effectively as nonsurgical treatment of localized fat deposits in abdomen, neck, arms and thighs [3]. PC has first a lipocyte-destroying effect and then a lipolytic action, which is active over 8weeks [4]. Despite being minimally invasive alternatives to liposuction, it causes localized and systemic side effects that usually appear 2-5days after application and include allergic reactions, tissue necrosis and body surface irregularities [5]. Nanotechnology-based delivery systems can protect drugs from degradation, reduce dose regimen, and enhance drug solubility. Vesicular system offers controlled drug delivery and increased drugs permeation through skin [6]. Transfersomes are metastable vesicles, sufficiently deformable to penetrate pores much smaller than their own size. They consist of phospholipids and one or more edge-activator [7]. Transethosomes are elastic ultra-deformable lipid vesicles containing high concentration of ethanol and edge-activators causing destabilization of the lipid bilayer and increases its flexibility [8]^. Accordingly, this work aims to introduce non-invasive dosage form of high patient compliance for efficient, safer treatment of localized obesity instead of the applied multi-injection regimen through application of nanotechnology in drug targeting.

Materials and Methods

Materials

Soybean PC, SDC, cremophorA25, cholesterol, chloroform and methanol HPLC grade were purchased from Sigma-Aldrich (Darmstadt, Germany). Potassium hydroxide, oleic Acid, tween 80, and ethanol were purchased from El-Nasr Pharmaceutical Chemicals (Cairo, Egypt). Trichloroacetic acid was purchased from Carl-Roth Company (Karlsruhe, Germany). Carbopol 934 was purchased from Arabic laboratory equipment (Cairo, Egypt).

Animals

Male Albino rats (50±5gm) were purchased from National Research Center, Giza, Egypt. The study protocol was conducted in accordance with the ethical procedures and policies approved by the Animal Care and Use Committee of Faculty of Pharmacy-German University in Cairo. All rats were maintained in the animal facility at 25±5ºC,12hours dark and 12hours light cycle.

Methods

Quantitative analysis of PC

A modified method was applied for PC determination. A Spectrasystem High performance liquid chromatography (HPLC) consisting of Spectrasystem pump P2000 and detector UV-3000 connected to Thermo C8 reverse-phase analytical column (250mm lengthx4.6mm internal diameter and particle size 5μm) (Thermofisher, UK). The mobile phase consists of acidified water at pH3.5 and methanol [9]. Gradient elution was performed at a flow rate of 1.5ml/min from 80% to 100% methanol in 40min (as shown in Table1). In–vitro calibration curve was constructed using concentration range (7.5-62.5µg/ml) of PC standard solution in ultrapure water. 50µl of prepared solution was injected into the HPLC. The flow rate was adjusted at 1.5ml/min at 20˚C and detection was carried at 205nm [10]. The assay procedures were validated in terms of linearity, precision, and accuracy (R=0.9981, LOD=2.5ng/ml;LOQ=6.5ng/ml; interday and intraday assay RSD<10%, accuracy≈99%). PC concentrations in the withdrawn samples were calculated with reference to the calibration curve of area under the curve of peak corresponding to PC concentrations (Insert Table 1).

Table 1: Gradient elution of PC using methanol and acidified water

Time (min)	Acidified Water (%)	Methanol (%)
0	20	80
40	0	100
45	0	100
46	20	80
55	20	80

Preparation of Phosphatidylcholine Vesicles

Preparation of Transfersomes

Transfersomes were prepared using the thin film hydration method. PC and surfactants were solubilized in chloroform-methanol (2:1respectively) [11]. The organic solvent was evaporated leaving a dry thin film using a rotary evaporator (Buchi, Switzerland) at 55˚C, 80rpm and 471bars. The film was hydrated with 5ml ultrapure water previously heated at 55˚C. The hydrated vesicles were then rotated using rotary evaporator at 80rpm and 55˚C for 1hour at normal atmospheric pressure [12]. The prepared vesicles were left at room temperature for 2hours for swelling then kept at 4˚C [13]. The aforementioned prepared vesicles were labeled(F).

Preparation of Transethosomes Hydrated with 20%Ethanol

Transethosomes were prepared using the thin film hydration method similar to that used for preparation of transfersomes. However, the hydration step was performed with 5ml of 20%ethanol. These vesicles were labeled (Fe).

Preparation of Transethosomes

Transethosomes were prepared using the solvent dispersion method. PC and surfactants were solubilized in 1.5ml of 20%ethanol [14] under vigorous stirring in tightly covered round bottom flask in a water bath at 30˚C. An aliquot of 3.5ml ultrapure water [15] was added slowly under continuous stirring. The nanosuspension was left at room temperature for 30min under continuous stirring. The prepared formulations were left at room temperature for 2hours then kept in at 4˚C^[13]. They were then labeled (Et).

D-Optimal Mixture Design Model

In order to investigate the different effects of the used ingredients for preparing the vesicles which are composed of PC together with a blend of surfactants, a D-optimal mixture design was conducted using the Design-Expert 7.0software [16]. The demonstrated independent variables were: the individual amounts of each of Cremophor, Sodium deoxycholate (SDC), Tween 80 and Oleic acid. The responses were: the particle size, polydispersity index and the %yield. Values of the dependent variables; particle size (P.S), polydispersity index (PDI) and yield percentage (%yield), were fed into the utilized software and equations linking the dependent and independent variables were produced. The composition of the prepared formulations for the full experimental design is shown in Table2. Three Models were conducted; P.S, PDI and %yield respectively (Insert Table 2).

Table 2: Composition of the prepared nanovesicles (mg)

Formula			PC (mg)	CHOL (mg)	Tween80 (mg)	Sodium deoxycholate (mg)	Cremophor (mg)	Oleic acid (mg)
Transfersomes prepared by thin film hydration method	Transethosomes prepared by thin film hydration method	Transethosomes prepared by solvent dispersion method
F 1	F 1e	Et 1	80	0	10	10	0	0
F 2	F 2e	Et 2	80	0	0	10	0	10
F 3	F 3e	Et 3	80	0	0	0	20	0
F 4	F 4e	Et 4	80	0	2.5	12.5	2.5	2.5
F 5	F 5e	Et 5	80	0	10	0	0	10
F 6	F 6e	Et 6	80	0	0	20	0	0
F 7	F 7e	Et 7	80	0	0	20	0	0
F 8	F 8e	Et 8	80	0	0	0	0	20
F 9	F 9e	Et 9	80	0	20	0	0	0
F 10	F 10e	Et 10	80	0	10	0	10	0
F 11	F 11e	Et 11	80	0	12.5	2.5	2.5	2.5
F 12	F 12e	Et 12	80	0	0	0	0	20
F 13	F 13e	Et 13	80	0	0	0	20	0
F 14	F 14e	Et 14	80	0	0	10	10	0
F 15	F 15e	Et 15	80	0	5	5	5	5
F 16	F 16e	Et 16	80	0	0	0	10	10
F 17	F 17e	Et 17	100	0	0	0	0	0
F 18	F 18e	Et 18	80	20	0	0	0	0
		Et 19	80	0	0	0	20	0
		Et 20	80	0	3	1	13	3
		Et 21	80	0	1	5	13	1
		Et 22	80	0	2	2	14	2
		Et 23	80	0	1	1	17	1
		Et 24	80	0	2.5	0	15	2.5
		Et 25	80	0	6	8	4	2
		Et 26	80	0	3	14	2	1

Characterizations of the Prepared Vesicles

Determination of Phosphatidylcholine %yield. PC %yield was determined using the ultracentrifugation method using cooling centrifuge (Hermle, Germany) where 1ml sample of each formulation was placed in a 1.5ml eppendorf [17] and centrifuged at 4˚C at a speed of 14000rpm for 2hours [18]. The supernatant was collected. The vesicles were washed with 0.5ml ultrapure water and recentrifuged for 2hours. The supernatant was separated and its total volume was detected. An aliquot of 50µl of the total supernatant was diluted and peak area was measured using HPLC at λ_max=205nm. The concentration was calculated according to the established calibration curve. Each sample was measured in triplicates and mean value was reported. %Yield was calculated as follows:

Particle Size, Polydispersity index and Zeta Potential (ZP) Measurement

An aliquot of a 1ml sample of the prepared formulation was ultra-centrifuged at 4˚C and 14000rpm for 2hours. Consequently, the supernatant was removed. The vesicles were washed with 0.5ml ultrapure water and recentrifuged for 2hours. The supernatant was removed and the vesicles were redispersed by vortexing for 10seconds. Afterwards, 0.35ml of the vesicles was diluted to 5ml with ultrapure water. P.S and surface charges of the nanovesicles were measured using Zeta-Sizer Nano-ZEN3600. The measurements were executed in triplicates for each sample and the average values were calculated [14;19].

Transmission Electron Microscopy (TEM)

The morphology of a dilute stock of selected nanovesicles was examined using electron transmission microscope TECNAI-G2 S-Twin (Netherlands) at 80KV after being stained with phosphotungstic acid [1].

Elasticity Test

The selected vesicles elasticity test was performed using the extrusion method^[20] where the nanosuspension was extruded through Micropore cellulose membrane filter of 0.22µm at a constant flow of 115L/min. Deformability was reported as the deformability index (DI) calculated by following equation [21]:

DI=j×(rv/rp)²   (Equation2)

Where, j is the suspension flow rate, rv the vesicle size and rp the membrane pore size [20].

Stability testing of the prepared nanovesicles

Stability tests were performed for the selected formulae stored at 4˚C for 6months. The stability was assessed by measuring %yield, ZP, P.S and PDI. Measurements were executed in triplicates for each sample and the mean values were calculated [22].

Preparation of the nano-phosphatidylcholine vesicular gel:

The selected nanovesicles were centrifuged at 4ºC, 14000rpm for 2hours. The supernatant was removed and samples were redispersed using 0.5ml ultrapure water. Nanovesicular gel with 2%carbopol 934 was prepared using triethanolamine, methyl and propyl parabens [23]. The prepared gel was stored at 4˚C.

Characterizations of the nanovesicular gel:

Physical examination

The developed gel was tested for color, transparency, homogeneity by visual inspection [24].

pH measurement

The pH of 1%aqueous solutions of the prepared gels was measured using pH-meter (Jenway, UK) [25].

Viscosity Studies

The gel apparent viscosity was measured using Visco-star plus Viscometer (Fungilab, Barcelona) at room temperature. Readings were taken after 5min. Measurements were executed in triplicates [25].

Spreadability Test

Spreadability was assayed by pressing 0.5gm of gel between two glass slides till no more spreading occurs. Four diameters, for each of the formed circle, were measured and the average diameter was calculated. The mean of triplicates of each formulation was used as comparative values for spreadability [26].

Drug content determination

Drug content of the gel was quantified. 250mg of the prepared gel was mixed with 10ml water thoroughly. The produced solution was centrifuged for 30min at 6000rpm using Hermle centrifuge (Wehingen, Germany). The supernatant was then filtered, 0.5ml of supernatant was diluted. Drug content was determined using HPLC. The concentration was obtained using the established calibration curve at a λ_max of 205nm [27].

Stability of the nanovesicular gel

Stability was studied after storage of the prepared gel at 4˚C for total periods of 3and 6months. The stability was assessed through measuring viscosity, pH and drug content. The measurements were executed in triplicate for each sample. Average values were obtained [22].

In-vivo studies on rats

Induction of obesity

Rats were randomly assigned to one of the two groups, the model group(n=42) or the control group(n=6). They were allowed free access to regular rat chow (the formula of low-fat diet) and tap water for 1week. The rats of control group were fed with rat chow. Other groups were fed with high-fat diet containing 70%lard. Meal administration continued for 4months. The criterion of successful induction of obesity was reaching 450±5gm body weight. Successful rats were divided into 4main groups (GpII to GpV). After obesity induction and during treatment period, all groups were fed with low-fat diet while model group was still fed with high-fat diet [28].

Treatment Groups

Adult male rats (50g±5) were divided into 5groups:

Group I: Control group (Allowed to low-fat diet without drug administration) and consists of 6rats

Group II: Model group (Allowed to high-fat diet without drug administration) and consists of 6rats.

Group III: Low-fat diet group (Allowed to high-fat diet and treated with low-fat diet without drug administration) and consists of 6rats.

Group IV: Market Treatment group (Allowed to high-fat diet and treated with low-fat diet & PC-market injection (Adipoforte^®)) and consists of 6rats.

Group V: Introduced dosage form Treatment Group (Allowed to high-fat diet and treated with low-fat diet & PC new topical dosage form) and consists of 24rats. It was divided into 4subgroups (Group Va, Vb, Vc and Vd) for each formulation of the 4different selected formulations (F4e, F11e, Et11 and Et20) respectively. Each subgroup consists of 6rats.

Treatment and drug delivery

According to Lu,2014 _[1], the abdominal hair was shaved and then intervened by drug application. Animals in the model group (GrpII) were massaged with the same amount of water on the abdomen in a clockwise direction. Animals in (GrpIII) were not treated with any massage or drug administration. The rats in the market treatment group (GrpIV) were injected subcutaneously with Adipoforte® at a dose of 0.85mg/day [29] for 8weeks. GrpV received PC new topical formulation at a dose equivalent to 0.85mgPC/day. Treatment was administered at the morning for 8weeks [30, 31]. Rats were weighed 2times/week before drug administration using electronic balance TE-612 (Sartorius AG, Germany) [1].

Skin irritation test

For skin irritation test, 36rats were divided into 3groups:

Group I: Control Group and consists of 6rats

Group IV: Market Treatment Group and consist of 6rats.

Group V: Introduced dosage form Treatment Group and consists of 24rats. It was divided into 4subgroups (Group Va, Vb, Vc and Vd) for each of the four different selected formulations (F4e, F11e, Et11 and Et20) respectively. Each subgroup consists of 6rats. Amount of gel equivalent to 0.85mg PC was applied to shaved area of group V (n=6 for each formula of prepared PC vesicular gel); same way control gel was applied to group I for the determination of irritation characteristics and hypersensitivity reaction on the skin. Group IV was injected with market PC injection (Adipoforte^®). The visual observation was carried out at regular interval of 10, 24 and 48hours [24]. The erythema and edema were scored as follows: none=0, slight=1, well defined=2, moderate=3, and 4 for severe erythema, edema and scar formation [32].

Dorsal fat percentage and total lipid content analysis

At the end of treatment, rats were weighed and sacrificed. Dorsal adipose tissues were removed and weighed (wet weight of dorsal fats) after excess blood and tissue fluids were dried by filter paper. Dorsal fat percentage (PDF) was calculated^[1].

The dorsal adipose tissue was digested in hot 30%KOH using homogenizer (Wiggenhauser, Germany) and then acidified. The produced homogenate was centrifuged for 2hours at 6000rpm. Total Lipid content was extracted with chloroform-methanol (2:1respectively) where organic phase was isolated and evaporated to dryness using rotary evaporator (Buchi, Switzerland). The total remaining lipid content was weighed [33].

Statistical Analysis

Data statistical analysis was performed with nonparametric one-way ANOVA test. Results were expressed as mean ±SD. All statistical tests were two-sided.

Results

%Yield Model for transfersomes (formulations code starting with F)

The obtained model was a quadratic one. By applying ANOVA test, it was nonsignificant(P=0.04) though with a desired nonsignificant lack of fit. All the linear mixture components: A, B and D were significant while C and other quadratic terms: AB, AC, AD, BC, BD and CD were nonsignificant. Accordingly, model reduction was carried out and ANOVA was reconducted. A significant model was obtained with a desired nonsignificant lack of fit. The results modeling showed r² of 0.700, adjusted r² of 0.55 and a predicted r² of 0.44. The predicted r² is in a reasonable agreement with the adjusted r² (Difference between them<0.2). The Box-Cox plot for power transforms demonstrated the approximate coincident of the current lambda (1) with the best lambda (1.14) lying within the confidence intervals (-0.17to2.99) [34]. The obtained contour plots are shown in Figure1 (Insert Figure 1).

Figure 1: Contour Plot demonstrating the effect of (a)Oleic acid, cremophor and Tween 80 (b)Oleic acid, cremophor and SDC on the %yield of PC-transfersomes

The obtained Model Equation obtained was:

%Yield=2.502*Tween80+2.451*SDC+3.486*Cremophor+4.387*Oleicacid (Equation4)

%Yield Model for transethosomes (formulations code starting with Fe)

The obtained model was a quadratic one. By applying ANOVA test, it was significant(P<0.0001) with a desired nonsignificant lack of fit. All the linear mixture components: A, B, C and D besides the terms AB, AC, AD, BC and BD were significant. The CD quadratic term was nonsignificant. Accordingly, model reduction was carried out and ANOVA was reconducted. A significant model was obtained with a desired nonsignificant lack of fit. The results modeling was successful as demonstrated by the values of r² (0.98), adjusted r² (0.96) and predicted r² (0.76). The predicted r² is in a reasonable agreement with the adjusted r². The Box-Cox plot for power transforms demonstrated the approximate coincident of the current lambda (1) with the best lambda (1.32) lying within the confidence intervals (0.41to2.61). The obtained contour plots are shown in Figure2 (Insert Fig 2).

Figure 2: Contour Plot demonstrating the effect of (a)Oleic acid, cremophor and Tween 80 (b)Oleic acid, cremophor and SDC on the %yield of PC-transethosomes prepared by thin film hydration

The obtained Model Equation obtained was:

%Yield Model for transethosomes (formulations code starting with Et)

The obtained model was a quadratic one. By applying ANOVA test, it was significant(P=0.0056) but with a non-desired significant lack of fit. All the linear mixture components: A, B, C and D besides the term AC were significant. The other quadratic terms: AB, AD, BC, BD and CD were nonsignificant. Accordingly, model reduction was performed. This time a significant model was obtained with a higher p-value for lack of fit but still significant. The modeling of the results was successful as demonstrated by the values of r² (0.81), adjusted r2 (0.74) though the predicted r² was low (0.31). The Box-Cox plot for power transforms demonstrated the approximate coincident of the current lambda (1) with the best lambda (1.33) lying within the confidence intervals (-0.15to2.96). The obtained contour plots are shown in Figure3.

Figure 3: Contour Plot demonstrating the effect of (a)Oleic acid, cremophor and Tween 80 (b)Oleic acid, cremophor and SDC (c)Oleic acid, Tween 80 and SDC on the % yield of PC-transethosomes prepared by solvent dispersion

The model equation obtained was:

%Yield=1.396*Tween80+3.229*SDC+3.798*Cremophore+2.567*Oleicacid0.279*Tween80*Cremophore            (Equation6)

Validation of experimental design

Eight new formulations (Et19-Et26) were chosen. The actual %yield, P.S and PDI were compared with the predicted values. For %yield model, the obtained values were comparable to the predicted counterparts, these results ensured the validity of the %yield model with a mean %bias of 7.4%. For P.S and PDI models, the overall mean was considered a better predictor of the response than the obtained models due to the obtained negative predicted r². Thus, these models are not reliable [35] and were considered as reported values.

Zeta Potential

ZP give indication about surface charge type and magnitude [36] which can affect both vesicular stability and skin-vesicle interactions^[8;37]. Vesicles showed highly negative charges ranging from -31 to -72.5mV for the prepared transfersomes (F), -24.8 to -53mV for the prepared transethosomes (Et) and ranging from -23.2 to -64.2mV for the prepared transethosomes (Fe). For the transethosomal formulations (Et21 to Et26), no significance difference was observed in their ZP(P>0.05).

Selection of the formula of choice

From the data shown in Table3, it was found that transethosomes (F4e, F11e, Et11 & Et20) showed SD<10% of the mean of the evaluated parameters indicating their reproducibility. Their P.S ranged from 200 to 480nm, so they can reach the skin subcutaneous layer and become entrapped. Besides, their ZP range is between -41.8mV and -53.2mV ensuring particle stability with reduced mutual aggregation. Moreover, they have PDI around 0.3 ensuring low variability. Their %yield ranged between 31.3% to 63.89%, on which the dose will be calculated. Consequently, F4e, F11e, Et11 & Et20 were selected as formulations of choice on which further studies were done.

Elasticity Test

The elasticity results are shown in Table4. The chosen formulations showed a high deformability index ranging from 70.8976±4.29 to 137.1707±6.14 (Insert Table 3 & 4).

Table 3: Characteristics of the selected vesicles

Table 4: Particle size and Deformability Index of the selected vesicles before and after extrusion

Formula Code	Particle Size (nm)		Deformability Index (DI)	Average DI ± SD
	Before Extrusion	After Extrusion
F4e	476.8 ± 3.72	176.73	74.2118	70.8976 ± 4.29
		174.6	72.43
		166.73	66.051
F11e	450.1 ± 2.06	235.6	131.887	137.1707 ± 6.14
		239	135.72
		246.13	143.905
Et11	239.8 ± 5.52	188.3	84.25	88.4467 ± 4.26
		197.6	92.77
		192.8	88.32
Et20	236.5 ± 4.99	189.6	85.41	84.26133± 2.72

Transmission Electron Microscopy

The morphology of the four selected formulations is shown in Figure4. The imaging analysis showed unilamellar vesicles possessing a thin lipid layer that is hydrated forming enclosed vesicular structure whose shape ranges from spherical to oval with some irregular shapes and black precipitates (Insert Fig 4).

Figure 4: TEM micrographs of formulation (a)F4e, (b)F11e, (c)Et11, (d)Et20

Stability test

The stability of the selected formulae was evaluated by macroscopic inspection and by measuring their P.S, PDI and %yield monthly and ZP every 3months for 6months storage at 4˚C. At room temperature, fungal growth appeared after the first month. An adequate stability of the selected transethosomes (F4e, F11e, and Et11) was observed with nonsignificant change regarding their P.S, PDI, %yield and ZP through the 6months of storage at 4˚C(P-value>0.05). Transethosome formula (Et20) showed nonsignificant differences regarding PDI, %yield and ZP throughout the 6months (P-value=0.4279, 0.4344, 0.4291, 0.4225, 0.4287 & 0.4247) but showed a highly significant change in P.S compared to P.S of original samples throughout the 6months (P-value=0.0009, 0.0002, 0.0091, 0.0077, 0.0068 & 0.0091).

Characterizations of nanovesicular gel

All prepared gels were translucent, smooth, and consistent in appearance with pleasant acceptable odor and without appearance of any clumps nor phase separation.

pH Measurement

The pH of all prepared gels was found to range from 8.24±0.36 to 8.78±0.14.

Viscosity Studies

The prepared gel viscosity ranged from 71254±4cps to 77183±3cps ensuring the successful preparation of the gel structure.

Spreadability Test

The prepared gel spreadability was measured in terms of average diameter of the spread circle. The longer the diameter, the better the spreadability [26]. Measurements lie between 3.43±0.14cm and 3.75±0.13cm indicating good spreadability properties.

Drug content determination

Drug content of PC was found to be 94.53±0.02%, 95.476±0.1%, 95.43±0.35%, and 96.875±0.1% for F4e, F11e, Et11 and Et20 gels respectively.

Stability Studies of gel

Nanovesicular gel color, consistency, pH, drug content and viscosity were evaluated after 3and 6months of storage at 4˚C [24]. The prepared gels were consistent with no signs of phase separation or deterioration.

In-vivo studies on rats

In–vivo studies of PC-vesicular gel formulations containing F4e, F11e, Et11 and Et20 were performed and results are presented in Table (5,6) and Figure (5–8).

Induction of obesity

As shown in Table5 & Figure 6(a), the body weights were similar among all groups before initiation of high-fat diet(P>0.05). At the end of 4months on high-fat diet, the body weight of obese rats (GpII-V) significantly increased compared to control group (GpI)(P<0.0001).

Table 5: Rats body weight changes before and after treatment

Rat Group	Initial body weight	Body weight before high fat diet	Body weight after high fat diet	Body weight after treatment	%weight loss
Control	51.3 ± 2.43	74.1233 ± 5.437	227.016 ± 5.937	297.08 ± 27.648
Model	50.92 ± 3.64	76.0775 ± 7.0175	451.865 ± 3.4027	462.917 ± 24.076
Diet only			451.95 ± 2.3036	418.833 ± 51.219	7.328
Injected			449.417 ± 3.11	397.33 ± 38.867	11.59
F4e			452.03 ± 3.1123	356.667 ± 62.67	21.097
F11e			451.383 ± 3.517	394.33 ± 23.157	12.64
Et 11			450.433 ± 4.9066	379.833 ± 34.649	15.6
Et 20			450.817 ± 3.414	394.5 ± 29.751	12.4

Skin irritation test

In the control (GpI) and treated (GpVa to Vd) groups, the erythema score was 0 and no irritation signs appear through the total examination period (Figure5(a) and (b) respectively). In grpIV, that was injected with Adipoforte^®, slight erythema with score 1 appear after 24hours in 50% of the group (Figure5(c)). After 48hours, a hard scar appeared with erythema score of 4 ((Figure5(d), (e) and (f)) forming hard nodules or lesion that disappeared after 3days (Insert Figure 5).

Figure 5: Irritation score zero in (a)control group (Gp I), (b)treated groups (GpVa-Vd), (c)Irritation score 1 in injected group (Gp IV) after 24hours, (d,e,f)Irritation score 4 in injected group (Gp IV) after 48hours

Treatment and drug delivery

As shown in Table5 & Figure6, the body weight of obese rats (GpVa) after obesity treatment showed nonsignificant difference compared to the control group (P=0.1647). In comparison to the model group (GpII), the body weight of the treated groups (i.e. GpVa and Vc) was reduced significantly (P<0.05) with %weight loss of 21.097% and 15.6% respectively. On the other hand, there was nonsignificant difference in weight reduction between the model group (GpII) and the group treated with diet only (GpIII)(P=0.5142) whose body weight decreases by 7.328%. There was nonsignificant difference in weight reduction between the model group (GpII) and the group treated with injection (GpIV)(P=0.0932). There was nonsignificant difference in weight reduction between the model group (GpII) and the group treated with prepared topical formulations containing F11e, Et20 (GpVb and Vd) (P=0.0686 & P=0.0698 respectively) whose body weight decreases by 12.64% and 12.4% respectively. There was nonsignificant difference between the diet group (GpIII) and treated groups (GpV)(P>0.05) (Insert Table 5& Fig 6).

Figure 6: ** P value ≤ 0.01 indicating significant difference
*** P value ≤ 0.001 indicating highly significant difference
**** P value ≤ 0.0001 indicating an extremely significant difference
(a)Body weight changes among treatment groups, (b)%weight loss changes among different treatment groups after end of treatment

Dorsal Fat Percentage and total lipid content

According to %PDF, a highly significant difference between the control group and other groups (P<0.0001) appeared as shown in Table6, and Figure7. The %PDF of the injected obese rats (GpIV) was nearly equal to that of the control group by the end of treatment (P>0.9999). On comparing model group (GpII) with the treated groups, the %PDF of the treated groups (GpIV and V) reduced significantly(P<0.0001). As shown in Table6 and Figure8, the total Lipid content of the treated groups (GpIII, IV and V) reduced significantly (P<0.0001) compared to model group. Meanwhile, there was a highly significant difference between the control group (GpI) and the model, diet and injected groups (GpII, III, and IV) (P<0.0001). Interestingly, the total lipid content of the control group (GpI) compared to the treated group with PC vesicular gel (GpV) showed nonsignificant difference (P>0.05). Total lipid content in rat group treated with diet only (GpIII)(4.834±0.403g) decreased to half that of the model group (GpII)(9.05±0.7319g). On comparing model group (GpII) with injected group (GpIV) and invented new dosage form group (GpV), total lipid content decreased in group IV and group V by 67.96% and 88.398% respectively reaching in the latter group a comparable value as that of control group (GpI) (Insert Table 6& Fig 7-8).

Table 6: Comparison of obesity parameters among groups

Rat Group	Rat weight (gm)	Fat tissue wet weight (gm)	%PDF	Total Lipid content weight (gm)
Control	297.08 ± 27.648	2.286 ± 0.2034	0.7754 ± 0.1029	1.552 ± 0.266
Model	462.917 ± 24.076	9.366 ± 0.6992	2.0218 ± 0.074	9.05 ± 0.7319
Diet only	418.833 ± 51.219	5.387 ± 0.707	1.3098 ± 0.2772	4.834 ± 0.403
Injected	397.33 ± 38.867	3.1 ± 0.4406	0.781 ± 0.0893	2.9 ± 0.5168
F4e	356.667 ± 62.67	1.218 ± 0.604	0.3305 ± 0.1151	1.103 ± 0.488
F11e	394.33 ± 23.157	1.294 ± 0.486	0.3245 ± 0.1104	1.102 ± 0.4028
Et 11	379.833 ± 34.649	1.099 ± 0.346	0.2845 ± 0.0693	1.019 ± 0.3044
Et 20	394.5 ± 29.751	1.169 ± 0.3592	0.292 ± 0.0708	1.094 ± 0.307

Figure 7: ns P value > 0.05 indicating no significant difference
**** P value ≤ 0.0001 indicating an extremely significant difference
(a) Changes in %PDF among different groups, (b)%PDF obtained for all treatment groups

Figure 8: ns P value > 0.05 indicating no significant difference
**** P value ≤ 0.0001 indicating an extremely significant difference
Changes in total lipid content among different treatment groups

Discussion

The contour plots obtained confirm the interaction effects of the used oils and edge-activators in increasing the %yield of the prepared transfersomes and transethosomes. For the three types of prepared vesicles, the area of high %yield lies between oleic acid and cremophor which indicates that by increasing their percentage, the %yield increases. Increasing the percentage of Tween 80 decreases the %yield demonstrated by the blue area close to its apex. For transfersomes and transethosomes prepared by thin film hydration method, increasing SDC decreases %yield demonstrated by the blue area close to its apex where increasing tween 80 and SDC causes lipid layer destabilization leading to reduced %yield [22, 38]. Also, increasing the concentration of some edge-activators beyond certain threshold leads to formation of micelles instead of vesicles causing solubilization of the phospholipids [19, 39]. This can be attributed to cremophor bulky structure which provides rigidity to the vesicles leading to higher P.S increasing %yield [40]. According to literature, increasing HLB value as in the case of cremophor [41] increases the P.S which can in turn increase %yield by increasing hydrophilicity, enhancing surface free energy^[39]. Increasing SDC, in transethosomes prepared by solvent dispersion method, increases %yield demonstrated by the yellow area close to its apex where SDC increases the whole lipid bilayer volume increasing P.S causing increase in %yield [42]. For good physical stability [43], ZP should not be less than -30 or +30mV [37] and on approaching -60mV [44], vesicles obtain an excellent physical stability through shelf life preventing aggregation [45]. This means that all formulations are stable except (F1e, F10e, F13e, Et3, Et10, Et13 and Et14). This can be attributed to the presence of edge-activators [8]; tween 80 was reported to cause decrease in ZP, although it is a nonionic surfactant [46] due to its oxyethylene part^[47]. Oleic acid was reported to produce negative ZP [6;48]. Using SDC produces high negatively charged vesicles due to the presence of cholate anions [47]. Although PC is zwitterionic compound with an isoelectric point (6-7), PC carried a net negative charge under experimental conditions of pH7.4 [12;39] due to the negatively charged phosphate group^[43;49]. For transethosomes, ethanol produces negative charges on vesicles [14;45]. These negatively charged vesicles enhances skin permeation of drugs [12]. Lipid bilayer elasticity affects permeation enhancing skin penetration^[47]. The edge-activator chemical structure affects vesicles deformability where flexible non-bulky carbon chain gives more fluidity to the membrane bilayer compared to bulky cyclic edge-activators [39]. Transethosomal formula (F11e) show the highest DI followed by Et11 then Et20 and finally F4e showing the least DI. This can be attributed to the high concentration (12.5%) of tween 80 with its highly flexible and non-bulky hydrocarbon chains [20] which aid in their squeezing along the stratum corneum and localization at high concentration in the deepest skin layers [47, 50]. Although Et11 and F11e have the same percentage of Tween 80, F11e has a higher ethanol volume (5ml) than Et11 (1.5ml) where increasing ethanol content increases lipid bilayer elasticity [8, 51]. Et20 showed lower DI compared to F11e due to high concentration of cremophor (12.5%) with its bulkier structure compared to Tween 80^[40]. F4e showed the lowest DI with the highest percentage of P.S change comparing P.S before (476.75±3.718nm) and after extrusion (172.69±5.27nm) whereas %P.S change increases, DI decreases. This can also be due to high concentration of SDC (12.5%) with its steroid-like structure [52]. The use of oleic acid and ethanol provides high elasticity [51]. The TEM micrographs show a highly recognized vesicles in the nanometer range which agreed with the size data obtained using dynamic light scattering (DLS) and ensures vesicle formation at the used concentrations of ethanol and edge-activators [8]. Deviation of particle shape from spherical form is due to lipid modification during sample drying for imaging [45] and being highly deformable [43, 47]. The slight change in size can be attributed to the samples drying prior imaging [53]. The appearance of black precipitates may be due to precipitation of phosphotungstic acid in hydrophilic core [53, 54]. The selected transethosomes (F4e, F11e, and Et11) adequate stability can be due to their high ZP [44, 45]. Transethosome formula (Et20) shows a highly significant change in P.S compared to freshly prepared samples throughout the 6months (P-value=0.0009, 0.0002, 0.0091, 0.0077, 0.0068 & 0.0091), however, it is still in the size range targeting skin subcutaneous layer (185-460nm) [1]. It was reported that high cremophor concentration causes physical instability [55] due to enhanced water penetration into vesicle increasing P.S upon storage [56]. The pH of the prepared gel lies in the physiologically accepted range of 5-9 [57, 58]. Gel viscosity affects the extrudability, drug release [27] and vesicles delivery onto or across the skin [45]. The prepared gel high viscosity, due to the presence of lipid vesicles [39], facilitates the retention of gel on the skin for better skin penetration. The prepared gel spreadability indicates that the gel is easily spread by low shear^[24] with uniform spreadability [26, 32]. Drug content results show homogenous dispersion of vesicles in gel^[39] indicating the suitability of method used for gel preparation [27, 59]. On comparing the original results and those obtained after storing gel for 3and 6months, nonsignificant change was observed in the above mentioned parameters (P-value>0.05) ensuring stability over 6months [59]. Average body weights after high-fat diet, shown in Table5 column4, shows about 50% increase compared to the normal control group (GpI) indicating the successful establishment of obesity model by feeding the animals with high-fat chow containing 70%lard [1]. In comparison to the model group (GpII), the body weight of the treated groups (GpVa and Vc) was reduced significantly (P<0.05) with %weight loss of 21.097% and 15.6% respectively indicating their ability to reduce body weight. On the other hand, there was nonsignificant difference in weight reduction between the model group (GpII) and the group treated with diet only (GpIII)(P=0.5142) whose body weight was decreased by 7.328% which is in coordinance with previous studies which stated that diet regimens failed to act on localized obesity [5]. Skin irritation test was conducted to assess the potential irritant effect of PC vesicular gel formulations [22]. The appearance of hard scar in rats injected with PC-market injection, confirms lack of patient compliance of injection lipolysis for treatment of localized obesity which is in accordance with previous experiments which reported that localized adverse effects were described as “very mild’(18.4%) or “mild”(39.2%) [60]. According to observed changes in body weight, %PDF and total lipid content observed, the topical application of PC vesicular gel, revealed the ability of newly prepared gel containing transethosomes (F4e, F11e, Et11 & Et20) to significantly decrease localized fat. This confirms the successful penetration of vesicles into the skin subcutaneous layer that can be attributed to the solvent action of ethanol, used in preparation of transethosomes on stratum corneum, in addition to the high deformability and malleability of these vesicles. This aids in their squeezing along the stratum corneum and localizing at high concentrations in subcutaneous layer producing their lipolysis effect^[15].

Conclusion

PC nanovesicular gel, containing transethosomes (F4e, F11e & Et11), can be used as effective non-invasive treatment for localized obesity as an alternative to multi-injections for mesotherapy.

Conflict of Interest

No conflict of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

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