3.1. Measurement of the pH Value
pH values of obtained hydrogels are presented in Table 2
The pH values of the hydrogels are close to the acidic character. The lowest pH value was observed for glycerol hydrogel as a base (F-1), whereas the highest for hydrogels containing adrenocorticotropic hormone, depending on ACTH concentration for concentration of 15 mg/g pH = 5.99 ± 0.028 (F-2) and higher for concentration of 20 mg/g, pH = 6.24 ± 0.014 (F-6). The results obtained allow us to state that adding ACTH to the glycerol hydrogel increases the pH value. On the other hand, the addition of albumin at any concentration causes a decrease in pH value in comparison with glycerol hydrogel with ACTH, the more albumin, the lower the pH value. The pH values of the obtained hydrogels are similar to the pH value of the skin (4.5–6.5), so they can be safely applied to the skin without causing irritation.
The pH value as well as temperature-dependent gelation conditions for bovine serum albumin (BSA-bovine serum albumin) and human albumin (HSA-human serum albumin) have been studied by Arabi et al., 2018 [29
]. The researchers confirmed that gelation of BSA and HSA can occur in a wide range of pH and temperatures (pH 1.0–4.3 and pH > 10.6 at 37 °C or pH 7.0–7.2 at 50–65 °C). Hydrogen ions required during PBSA hydrogel formation (electrostatically (pH) triggered BSA hydrogels) can be neutralised so that product obtained has a physiological pH of 7.4. PBSA hydrogel remains stable for up to three months [30
3.2. Corticotropin Release Study from the Hydrogels
For studies, the release of ACTH from the obtained hydrogels, USP 2, Enhancer cells were used due to the possibility of release into a smaller volume of acceptor fluid. It is believed that the release of the active substance is determined by the properties of the membrane through which the substance penetrates into the acceptor environment. Thus, depending on the properties of the therapeutic substance, the drug formulation and the route of its administration, the release study can be performed in different fluids. Phosphate-buffered sodium chloride, pH 7.4, was used as the acceptor fluid.
In the release investigation, it was observed that from hydrogel of 15 mg/g, ACTH 2.28 ± 0.05 mg/cm2
was released (Figure 2
A), whereas from hydrogel of 20 mg/g ACTH 1.2 times more corticotropin was released −2.79 ± 0.15 mg/cm2
compared to hydrogel of 15 mg/g ACTH (Figure 2
B). However, referring to the fraction of dose placed on the cellulose membrane, increasing the dose from 15 to 20 mg/g (1.33 times) did not result in a corresponding increase in the amount of ACTH released. The amount of corticotropin released was 58% for 15 mg/g and 53% for 20 mg/g. 1.1 times more ACTH was observed from the lower concentration hydrogel. The addition of albumin in a 1:1 ratio to ACTH significantly increased ACTH release at the lower concentration of 15 mg/g by 1.4-fold, from 2.28 ± 0.05 mg/cm2
to 3.11 ± 0.19 mg/cm2
. In the case of a higher concentration −20 mg/g, initially albumin decreased the release of ACTH from the hydrogel, only after 180 min a positive effect of albumin on the increase in released ACTH was noted. The released ACTH increased from 2.79 ± 0.15 mg/cm2
to 3.18 ± 0.09 mg/cm2
, but the difference was not statistically significant.
The Weibull method (p
< 0.05) showed statistically significant differences in ACTH release profiles from the obtained hydrogels (p
< 0.05). The regression coefficients are presented in Table 3
. Correlation coefficients for pharmacokinetic models of ACTH release from hydrogels are also presented in Table 3
. In the present study, a time interval of 15 min to 360 min was to model the drug release rate. The drug permeation rate and applicability of the Higuchi model for all hydrogel formulations of peptide hormone was inferred from an R2
value of approximately 0.99 for all formulations according to the Higuchi model. For calculation of ACTH release rate from the hydrogel, the Higuchi model was used (Table 4
The most advantageous formulation was hydrogel with lower concentration of ACTH (15 mg/g), with addition of albumin in ratio 1:1 (15 mg/g), because albumin significantly increased the amount of released ACTH and for this preparation, the process of penetration through the natural membrane which is porcine skin was examined.
Tazhbayev et al. produced urea- and cysteine-stabilised HSA nanoparticles and incorporated anticancer drugs into them. Urea, as a natural denaturant, expands the biopolymer molecule and facilitates cysteine access to the molecule to interact with protein molecules. The performance of nanostructures depends on various factors, especially the concentration of precursors (urea, albumin, and cysteine). Optimum levels of particle size, polydispersity index, zeta potential and particle capacity were obtained at concentrations of albumin 20 mg/mL, cysteine 0.01 mg/mL and urea 10 mg/mL. This arrangement of nanoparticles with an initial HSA concentration of 20 mg/mL was relatively stable over time, and analysis of samples over two days showed neither particle aggregation nor destruction of the biopolymer structure. The particle size was kept constant [31
]. Therefore, values of 15 mg/g and 20 mg/g were chosen as the working range for the initial albumin concentrations. This range of albumin concentrations was also suggested in the study due to the stability of this protein. Although no nanostructures were produced here, it was important that the particles did not change their size in the hydrogel during storage. The addition of albumin in stoichiometric amounts 10 and 100 times smaller did not yield the expected results. [31
3.3. Corticotropin Permeation Study from the 1.5% Hydrogel with Addition Albumin in Stechiometric Ratio 1:1 through Porcine Skin
The profile of the permeation process of ACTH caused by albumin is shown in Figure 3
. Table 5
shows regression coefficients for individual kinetic models of the permeation process and the permeation rate.
It was reported that corticotropin (ACTH) penetrates to a more significant extent from semi-solid formulations with lower concentrations. Three concentrations were tested, and it was found that the highest amount of ACTH permeated from hydrogel of 1.5%–15.07 ± 2.71% and the lowest from hydrogel of 2.5%–6.95 ± 0.54% [12
]. The higher the concentration of corticotropin in the hydrogel, the lower the skin permeation.
In the present study, it was observed that the effect of albumin on ACTH release from the resulting hydrogel was greater at the lower concentration, i.e., 1.5%. For a concentration of 2%, albumin did not significantly increase the amount of ACTH released.
Therefore, the permeation of ACTH through the porcine skin from hydrogel with a concentration of 1.5% ACTH and an ACTH to albumin ratio of 1:1 was studied. It was observed that an average of 40.76 ± 3.51% ACTH permeated from 1.5% hydrogel. Compared to the amount of penetrated ACTH in the previous report of Siemiradzka et al. −15.07 ± 2.71% [12
], an increase in the amount of penetrated ACTH was observed about 2.7 times. Thus, the rate of penetration is directly influenced by the amount of ACTH penetrating, which can be expressed in mg/cm2
(5.29 ± 0.48 mg/cm2
) or as a part of the dose applied to the skin in % (30.5%). An increase in ACTH permeation under the in-fluence of albumin permeation per unit area was found from 5.29 ± 0.48 mg/cm2
(30.5%) for ACTH alone to 7.27 ± 0.77 mg/cm2
(41.92%) for hydrogel containing ACTH and albumin in the ratio 1:1 of 15 mg/g hydrogel (p
< 0.05). If albumin increases the amount of ACTH permeated through the skin to 7.27 ± 0.77 mg/cm2
(up to 41.92%-about a 1.4-fold increase), the permeation rate also increased (Table 5
) 1.4-fold from 0.139 ± 0.008 to 0.192 ± 0.020 mg/cm2
This can also be demonstrated by measuring the area under the AUC permeation curve. For the hydrogel containing 15 mg/g ACTH, the AUC was 5623.756, while in the presence of 1:1 albumin, the AUC was 7736.197, making a 1.4-fold increase in the area under the curve Table 5
). For comparison, a study on the penetration of albumin from the hydrogel was also performed. It was found that albumin itself penetrated less than ACTH and ACTH together with albumin (0.99 ± 0.37 mg/cm2
). Binding to plasma proteins protects compounds from oxidation, reduces their toxicity and prolongs their half-life; drugs that are strongly bound to plasma proteins often show low first-pass metabolism [32
Increasing the efficiency of skin permeation of various drugs can be aided by various techniques for conducting transport substances into nanoparticles. Gaurav et al. prepared transferosomes also named ultra-deformable liposomes. These deformable vesicles can transport drug through the skin, which is the largest human organ with a total mass of 3 kg and an area of 1.5–2.0 m2
. The size of this organ favours efficient delivery of both low and high molecular weight drugs through transferosomes. The prepared transferosomal formulations were evaluated for uptake efficiency, stability and skin penetration ability. It was found that ultra-deformable liposomes can be used to deliver various drugs, e.g., analgesics, anaesthetics, corticosteroids, anticancer drugs, sex hormones, insulin, proteins including albumin [34
In percutaneous administration, the corneal layer of the epidermis tends to impede the absorption of drugs through the skin. However, albumin nanoparticles can bypass this barrier by delivering them through hair follicles. Additionally, nanoparticles can be retained in hair follicles for a much longer period of time. This would enable systemic absorption and local action as well as possible permanent release [35
At present, nanoparticles show great potential as carriers of new drugs in percutaneous drug delivery. Smaller nanoparticle sizes can ensure a tight contact with the stratum corneum and enhance skin permeation. The advantage of using such colloidal carriers is to protect sensitive drugs from destruction and to control the rate of drug release from such carriers. Nanoparticles have dimensions ranging from 1 nm to 1000 nm [36
]. Currently, researchers’ interests are focused on polymeric nanoparticles due to their stability and ability to specifically modify the surface [40
]. A significantly higher permeation flux of aceclofenac (0.0681 ± 0.0008 g/cm2
< 0.05) was confirmed from a Carbopol 940 gel containing chitosan, albumin and aceclofenac nanoparticles than for a commercially available aceclofenac gel (0.0316 ± 0.0004 g/cm2
/h). Aceclofenac is a non-steroidal anti-inflammatory drug (NSAID) with a short half-life (4 h), used for the treatment of pain and inflammation [41
]. Shokri and Javar in turn studied the strengthening effect of zinc oxide and calcium phosphate nanoparticles on the total penetration of albumin through a part of a full mouse skin cut from the back of the mouse. The enhancing effect of albumin permeation under zinc oxide nanoparticles was stronger, whereas it was faster under calcium phosphate nanoparticles. The most albumin penetrated in the presence of zinc oxide and it was reached in 1.5 h (40.2 ± 3.6 mg), while in the presence of calcium phosphate maximum was achieved in 1 h, and the amount permeated was 33.8 ± 5.5 mg [44
3.4. Effect of Albumin on the Rheological Properties of Prepared Hydrogels with ACTH
In the present study, the viscosity of prepared hydrogel formulations was determined at 32.0 ± 0.5 °C at two shear rates: 15 s−1
and 30 s−1
. The examined viscosity and shear stress values are presented in Table 6
A decrease in viscosity with increasing shear rate was observed in all tested formulations. For shear rate D = 15 s−1, the highest value of structural viscosity was observed for the hydrogel with higher ACTH concentration (20 mg/g; 9.20 ± 0.17 Pa·s), while the hydrogels with lower ACTH concentration-F-2 (7.59 ± 0.19 Pa·s) showed lower viscosity. Thus, the addition of ACTH increases the viscosity of the tested hydrogels. The addition of 15 mg/g ACTH causes an approximately 1.3-fold increase in viscosity, while the addition of 20 mg/g ACTH causes a 1.5-fold increase in viscosity for both shear rates (D = 15 s−1 and D = 30 s−1). Albumin, in turn, causes a decrease in viscosity, the more albumin, the greater the decrease in viscosity. Albumin added to hydrogels in a 1:1 ratio to ACTH causes a decrease in viscosity by about 1.6 times at both shear rates (D = 15 s−1 and D = 30 s−1) at a concentration of 15 mg/g and by about 1.5 times at a concentration of 20 mg/g. As the addition of albumin was reduced, the viscosity gradually increased compared to hydrogels with ACTH without albumin.
The relationship between shear rate and viscosity is shown in Figure 4
A,B. The study was carried out in the range D = 5–200 s−1
All hydrogels showed an inversely proportional relationship between viscosity and shear rate or stress, which indicates the shear properties of these hydrogels. This observation indicates that the hydrogels can be easily applied to the skin each time a different shear rate is applied.
The relationship between shear rate (γ) and stress (τ) is shown in Figure 5
A,B. With increasing shear rate of hydrogels, the stress values increased. As the shear rate decreased, the hydrogel structure was restored. Weak bonds between the molecules of the system were ruptured, while after decreasing the shear rate, the bonds between the molecules were regenerated and the hydrogel structure was restored. The flow curves for all hydrogels were concave to the horizontal axis. This indicates a pseudoplastic character, which allowed the hydrogels to return to their original rheological state.
The relatively small area formed between ascending and descending curves indicated good regeneration of the systems (Figure 6
A,B). A system with good regeneration could increase adhesion on the skin and thus maintain a better therapeutic effect.