Selective anti-cancer activity against melanoma cells using 3-O-acetyl-β-boswellic acid-loaded 3D-Printed scaffold

Abstract This study aimed to develop a local 3 D-printed bioactive graft using poly-caprolacton (PCL) as a drug carrier and 3-O-acetyl-β-boswellic acid (β-ABA) as an anticancer compound. β-ABA-loaded 3 D-printed scaffold was fabricated and physically characterized. The results indicated more desirable mechanical and physical properties of the β-ABA-loaded PCL mat in comparison with the PCL scaffold. Following sustained release of β-ABA, the β-ABA-loaded PCL scaffold revealed selective cytotoxic activity against melanoma cells, while the PCL + ABA with the bolus delivery of β-ABA was toxic against fibroblast cells. Followed by the induction of apoptosis in melanoma cells at the gene level, the result of the western blot showed that the β-ABA-loaded scaffold significantly up-regulated P53 and down-regulated BCL2, with an increment in the ratio of Bax/BCL2. The selective anti-cancer properties of β-ABA-loaded 3 D printed scaffold against melanoma cells indicated that this scaffold could be potentially used as a bioactive graft to improve the melanoma treatment. Graphical Abstract


Introduction
Melanoma is known as the most aggressive type of skin cancer because of its high rate of metastasis and resistance against the treatment (Coit et al. 2009). In order to treat melanoma cases, surgery is recommended as a gold standard method to eradicate the tumor (Bhatia et al. 2009). Due to the high risk of reappearance of the tumor, post-operational therapies such as chemotherapy and radiotherapy are routinely applied (Bhatia et al. 2009). Despite the advances of the surgical techniques and the treatment, the prevention of tumor recurrence in the local tissue remains a challenge. Therefore, the optimal bioactive skin graft that speeds up the healing of the cutaneous surgical wound and prevents the recreation of the tumor in the local tissue is considered as the gold standard post-surgical treatment (Uddin et al. 2020).
The local drug delivery scaffolds to treat different tumors have been widely explored, and in the case of melanoma tumor, the local treatment is more workable for clinical studies because of its accessibility, non-invasive implantation, and better target efficiency (Liu et al. 2018). The local delivery systems can provide sustained release of drug with less toxicity, and better efficacy by avoiding excessive circulation in comparison with the conventional chemo-therapies (Liu et al. 2018). These bioactive scaffolds with the capability of local drug delivery would be composed of either synthetic polymers, that is, poly-caprolacton (PCL), and poly (lactic-co-glycolic acid) (PLGA) or natural polymers such as alginate, and chitosan (Tiwari et al. 2012). 3 D-printing technology allows fabricating predesigned multilayered drug-incorporating porous constructs with the capability to release the drug in a sustained manner (Evans et al. 2021). Among the various 3 D-printing methods, the extrusion-based solution printing (ESP) provides a better platform to load the drug into a wide variety of polymers with high loading efficiency, sustained release manner, and good stability (Kjar and Huang 2019).
The medicinally important triterpenic acids known as boswellic acids including boswellic acid (BA), 11-keto-b-boswellic acid (KBA), 3-O-acetyl-b-boswellic acid (b-ABA), and 3-O-acetyl-11-keto-b-Boswellic acid (AKBA) are isolated from resins of Boswellia trees (Huang et al. 2000). Boswellic acid derivatives have shown biological activities that make them potent to treat various cancers and inflammatory diseases. Despite to the strong cytotoxic properties of AKBA, ABA revealed more cytostatic properties rather than cytotoxic which make it potent to be used as a chemo-preventive agent (Kumar et al. 2016;Schmiech et al. 2019). Additionally, b-ABA showed a more selective anti-cancer impact because of less toxicity against normal cells, while it caused DNA fragmentation in melanoma cells (Kumar et al. 2016).
The complementary effect of b-ABA against melanoma and fibroblast cells is required to demolish cancer and accelerate the wound healing, in order to treat melanoma. It is also necessary to find an appropriate dose of b-ABA, which targets melanoma cells and has minimal toxicity against fibroblast cells. Long-term sustained dynamic release of -ABA is favourable for delivering a sufficient dose of medication while also preventing excessive drug accumulation in cells. In this study, b-ABA-loaded 3 D-printed PCL scaffold was designed as a local delivery skin patch with an apoptosis effect on the melanoma cells and a minimal toxicity against fibroblasts.

Results and discussion
The structure of b-ABA was determined in our previous studies (Rehman et al. 2018;Ur Rehman et al. 2020).
The light and scanning electron microscopic images of PCL and b-ABA-loaded PCL scaffolds were stated in Figure S1(A) and S1(B, C), respectively. Both scaffolds' structures have represented integrity, porosity, and interconnectivity.
As shown in Figure S1(D), the average diameter of the fibers in b-ABA-loaded PCL scaffold was significantly smaller than that of PCL scaffold, indicating finer fibers in b-ABA-loaded PCL scaffold. The smaller fiber's diameter is more favorable in skin tissue engineering because it provides more specific area for fibroblasts attachment. Moreover, pore size is important criteria which modulates nutrient/waste exchange and cell migration (Boekema et al. 2014). Figure S1(E) revealed that the average pore size of the -ABA-Loaded PCL was 734.4131.7 m, which is larger and more uniform than the pore size of the PCL scaffold (665.4180 m). Overall, compared to the bare PCL scaffold, b-ABA loading provided better morphology and a more uniform structure providing a suitable environment for proper proliferation of fibroblasts.
The crystalline properties of PCL and b-ABA-loaded PCL scaffolds were evaluated by XRD. As illustrated in Figure S2(A, B), the corresponding peaks of PCL were detected in both scaffolds around 2h ¼ 21.7 , 24.0 , and 22.3 ; reflecting (110), (200) and (111) crystal planes, respectively (Zhang et al. 2019). According to the XRD analysis (Table S1), both crystallinity index and crystallite size of ABA-loaded PCL scaffolds were more than those of PCL scaffolds. Apparently, this difference may be due to the crystalline structure of loaded ABA (Rajnikant et al. 2001) (Figure S2(C)). Due to the small amount of loaded ABA, the specific peaks of ABA could not be distinguished in the XRD pattern of the ABA-loaded PCL scaffold, but the ABA did improve the scaffold's crystallinity index and crystalline size.
The degradation, water absorption, and mechanical behavior of scaffolds have been represented in the supplementary material ( Figure S3 & S4 and Table S2). The cumulative release percentage of b-ABA from b-ABA-loaded scaffold is illustrated in Figure  S5(A). This result showed a slow and prolonged release of b-ABA within 14 days. Although an increasing trend in the release percentage of the b-ABA was observed between day 1 and 14, the rate of this increment was slow, probably because of the low solubility of b-ABA in aqueous buffers. This prolonged release profile of b-ABA from 3 D-printed PCL scaffold can be due to the provided specific area in the scaffolds geometry. The drug release rate would decrease as the specific area reduces (Lim et al. 2018). The sustained delivery of b-ABA would allow the drug to release gradually while this is not possible in the bolus deliver. Therefore, it is suspected that a prolonged delivery of b-ABA would be more economical and effective.
Fibroblast and melanoma cells were applied to evaluate the biocompatibility and anti-cancer activity of scaffolds. Figure S5(B) represents the normalized viability of treated cells compared with the negative control (TCP). The TCP þ b-ABA group indicated the least normalized viability (7.80%) of fibroblasts due to the bolus delivery of b-ABA, while PCL scaffolds represented the maximum normalized viability (54.07%) demonstrating the adequate biocompatibility of PCL scaffold. The viability of fibroblasts seeded on b-ABA-loaded PCL was significantly higher than PCL þ b-ABA, demonstrating the superiority of b-ABA sustained delivery in comparison with the b-ABA bolus delivery. Both PCL þ b-ABA and b-ABA-loaded PCL scaffolds showed lower viability in melanoma cells when compared to the PCL scaffold, proving the toxicity of ABA to these cells. There was no significant difference between the anti-cancer activity of b-ABA-loaded PCL and PCL þ b-ABA, whereas b-ABA-loaded PCL represented less toxicity against fibroblasts in comparison with PCL þ b-ABA.
Live and dead assay was applied to confirm the previous results. As shown in Figure S6(A, B), the green particles represent live cells while the red ones represent dead cells. PCL, as the bared scaffold, allowed fibroblast to grow and expand perfectly in 3 D-printed PCL structure, indicating that PCL 3 D-printed scaffold is a suitable extracellular matrix for fibroblasts. PCL with the bolus delivery of b-ABA almost killed all of the fibroblasts and melanoma cells, whereas sustained delivery of b-ABA in b-ABAloaded PCL only killed the cancer cells and not fibroblasts. Furthermore it has been demonstrated that the effect of b-ABA is time-dependent, which accounts for less effect of b-ABA in the sustained dynamic release compared to the static culture (Kumar et al. 2016). These encouraging findings suggest that the PCL scaffold loaded with b-ABA may be a promising skin graft for the treatment of melanoma. As shown in Figure S7(A), the expression of P53 in b-ABA-loaded scaffold groups was significantly higher than the one with bolus delivery. BCL2, as an anti-apoptosis gene, was down-regulated in the PCL þ b-ABA or b-ABA-loaded PCL seeded cells. The sustained delivery of b-ABA in b-ABA-loaded PCL was responsible for significant reduction in the expression of BCL2 in comparison with either PCL þ b-ABA or PCL. The results suggested that b-ABA-loaded scaffold induced apoptosis in melanoma cells, which might be mediated through overexpression of P53 and down-regulation of BCL2.
BCL2 protein expression in melanoma cells seeded on b-ABA-loaded PCL was less than that in TCP and PCL groups ( Figure S7(B, C)). Furthermore, over-expression of P53 protein was observed in the b-ABA-loaded scaffold group compared to the TCP, indicating that the b-ABA-loaded scaffold has superior anti-apoptotic behaviour. On the other hand, the PCL group represented a significant decrease and increment in the expression of BCL2 and P53 compared to the TCP, respectively. In parallel with these results, Syrovets et.al. have shown that b-ABA diminished the expression of the BCL2 protein in prostate cancer cells (Syrovets et al. 2000). Moreover, Bax can act as an apoptotic activator by creating a heterodimer with BCL2. The significant overexpression of Bax protein in comparison with the TCP has been represented in Figure  S7(C). The ratio of Bax/BCL2 expression level is considered as an important representative of the susceptibility to apoptosis (Vucicevic et al. 2016). As shown in Figure (S7D), the ratio of Bax to BCL2 in the ABA-loaded PCL group was significantly higher than TCP and PCL (p < 0.01). The increased ratio of Bax/BCL2 expression levels in the ABAloaded PCL group suggested superior apoptosis. Therefore, the controllable release of b-ABA may robust the apoptosis in melanoma cells.

Experimental
See the supplementary material.

Conclusions
In conclusion, a bioactive skin graft with selective anti-cancer activity against melanoma cancer cells has been developed. A uniform porous 3 D-printed PCL scaffold loaded with b-ABA as an anti-cancer agent was fabricated with improved physical and mechanical properties. The prolonged delivery of b-ABA had the maximal anti-proliferative activity against melanoma cells and the minimal cytotoxicity affect against fibroblasts. This important result may help in the treatment of melanoma cancer while also accelerating tissue regeneration. Finally, the mRNAs and protein expression results confirmed the superior apoptosis effect of the bioactive scaffold against melanoma cells. This work provides a platform for the development of bioactive skin grafts for melanoma local treatment and tumor resection postoperative care. Further preclinical studies are warranted to propose this bioactive scaffold as a promising graft for improving melanoma treatment.