SRB Assay for Cell Proliferation

To study the cytocompatibility of the synthesized (homo)- and (co)oligoesters with biosystems, their effects on cell growth were measured using sulforhodamine B staining. The HaCaT cell line was derived from normal human epidermis and the cells underwent spontaneous immortalization during successive passages [30]. These cells are not tumorigenic and they retained some properties of normal keratinocytes such as contact inhibition of proliferation or the ability to differentiate [31]. Therefore, the HaCaT cell line has become a popular model for testing dermal drugs and cosmetics. In the culture flask, this cell line grows as a monolayer of densely packed cells (Figure 6). The substances tested in our study were applied over a relatively wide concentration range (1–100 μg/mL). The concentration range of p-anisic acid (and its conjugates) was set up based on data referring to the biological

activities of that compound. It has been shown to influence the enzymatic activity of proteasome and lysosomal cathepsins at such low concentrations as 5 μM (0.76 μg/mL) [32]. Inhibition of tyrosinase activity was detectable from 0.1 mM (15 μg/mL), with an IC50 value 0.6 mM (91 μg/mL) [33]. Antiproliferative activity of p-anisic acid against HepG2 cancer cell line was observed for the concentration range of 1–100 μM, with IC50 coming to 25 μM (3.8 μg/mL) [34]. On the other hand, we consider such concentrations as achievable in the epidermis, taking into account possible cosmetic formulations, as well as release and absorption rates [23].

**Figure 6.** Phase-contrast micrograph of HaCaT keratinocytes in culture flask (original magnification: ×100).

As shown in Figure 7A, (p-AA-CH2-HP)n did not exert any detrimental effect on keratinocyte proliferation. In fact, in treated wells, average cell numbers were greater than in the control. The statistical analysis revealed significant differences in cultures treated with (p-AA-CH2-HP)n at concentrations of 10 and 30 μg/mL. As shown in Figure 7C, free p-anisic acid exerted a significant cell growth promotion effect at the concentration of 100 μg/mL. Mechanisms responsible for that effect are unknown as only scant information exists on the p-methoxybenzoic acid impact on human cell functions. It has been previously suggested that the conjugates of drugs and oligo-(R,S)-(3-hydoxybutyrate) (OHB) are more efficiently taken up by cells, compared with non-conjugated molecules [35]. More efficient accumulation inside cells would explain the ability of our (homo)oligoester to stimulate keratinocyte proliferation at lower concentrations, compared with the free acid.

A pattern of keratinocyte response to [(p-AA-CH2-HP)x-co-(HB)y] was slightly different (Figure 7B). That is, (co)oligoester increased cell proliferation at concentrations of 3, 10, and 30 μg/mL, but inhibited cellular growth at the highest dose (100 μg/mL). In our previous research [23], we demonstrated some growth inhibitory properties of oligo(3-hydroxybutyrate) carriers in HaCaT cells. Zawidlak-Wegrzynska et al. [35] demonstrated that conjugation of ibuprofen with OHB increased its antiproliferative effects in colon cancer HT-29 and HCT 116 cell lines. OHB oligomer alone also exhibited some growth inhibitory action, though at relatively large concentrations. Oligo(3-hydroxybutyrate) loaded into liposomes reduced the growth of L929 cells and induced cell death and cell cycle arrest at the G1/G0 phase [36]. On the basis of these observations, we suggest that the presence of HB units in the [(p-AA-CH2-HP)x-co-(HB)y] chain and somewhat greater molecular mass could possibly favour the revealing of its antiproliferative activity in the present study. The mechanism underlying the antiproliferative effect of OHB is currently unknown. However, it is worth pointing out that 3-hydroxybutyrate dose-dependently decreased proliferation of the human kidney HK-2 cell line [37]. It was accompanied by cell cycle arrest at the G0/G1 phase and increased p21WAF1 as well as p27kip1

protein expression. These changes were mediated by oxidative stress, Smad3, and TGF-β. It has been shown that mammalian cells or body fluids (as serum) are able to hydrolyse dimers and trimers of 3-hydroxybutyrate owing to the action of enzymes, for example, carboxylesterase [38]. Therefore, 3-hydroxybutyrate could mediate the growth inhibitory effects of [(p-AA-CH2-HP)x-co-(HB)y]. After all, mild inhibition of HaCaT cell growth was seen solely at a very high concentration of the studied (co)oligoester, which allows considering it biocompatible. In practice, it is advisable to carefully adjust its concentration in cosmetic preparation to obtain an optimal effect on epidermal cells.

**Figure 7.** Growth of HaCaT cells in the presence of (**A**) (p-AA-CH2-HP)n; (**B**) [(p-AA-CH2-HP)x-co-(HB)y]; and (**C**) p-anisic acid. Each bar represents the mean ± SD; \* *p* < 0.05 versus the control group (analysis of variance, ANOVA); C+—positive control.

#### **4. Conclusions**

With regard to likely applications of the developed (p-AA-CH2-HP)n and [(p-AA-CH2-HP)x-co-(HB)y] (co)oligoesters in the area of biomaterials, especially in cosmetology, comprehensive in vitro cytotoxicity tests as well as hydrolytic degradation under laboratory conditions were performed.

The application of ESI-mass spectrometry to characterize aqua-soluble hydrolytic degradation products of (p-AA-CH2-HP)n oligoesters and [(p-AA-CH2-HP)x-co-(HB)y] (co)oligoesters allowed us to determine their molecular level chemical structure and helped us to confirm the release of bioactive p-AA from the developed systems.

Biological comparative in vitro studies showed that the synthesized (co)oligoesters were non-toxic and were well tolerated by the HaCaT cells. Both (homo)- and (co)oligoesters exerted a beneficial effect on keratinocyte growth, especially at moderate concentrations. The highest concentration of the (co)oligoester caused only a mild inhibition of cell proliferation. The presented study shows the potential of the developed (homo)- and (co)oligoesters as novel controlled release and delivery systems for future applications in the cosmetics industry.

**Author Contributions:** Conceptualization, G.A. and M.M.M.; Methodology, M.M.M., G.A., A.O., and M.M-K.; Formal Analysis, M.M.M., M.Z., and A.O.; Investigation, M.M.M., M.Z., A.O., and M.M.-K.; Writing—Original Draft Preparation, M.M.M., G.A., and A.O.; Writing—Review & Editing, G.A. and M.M.M.; Supervision, G.A. and M.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 872152, project GREEN-MAP.

**Acknowledgments:** The authors are grateful to the late Tomasz Balakier for the synthesis of monomer (p-AA-CH2-PL), conducted under the guidance of Janusz Jurczak from the Institute of Organic Chemistry, Polish Academy of Sciences.

**Conflicts of Interest:** The authors declare no conflict of interest.
