| Abstract | So far, systemic administration of free chemotherapeutic drugs has been the main delivery route in cancer patients despite its several drawbacks. Besides the adverse side effects, higher doses of these drugs are required for them to reach the tumour sites which eventually may lead to the development of drug resistance and limit the effectiveness of these drugs. Therefore, there is a need to develop drug delivery vesicles to overcome such problems and carry drugs to the desired sites and hence maximise their therapeutic index while reducing their cytotoxic effect.
Liposomes, as drug carriers have been used for delivering several chemotherapeutic drugs and have shown some success in treating certain cancers. By encapsulating anticancer drugs within lipid layers or aqueous core, liposomes offer a secure platform for targeted delivery. Thymoquinone (TQ), a component of Nigella Sativa has been shown to have anti-inflammatory, antioxidant and anticancer properties for different types of cancers. Although it has been shown to induce cell apoptosis for cervical cancer cells in vitro, its anti-HPV effect and the mechanism of its action has not been fully investigated.
In addition, its hydrophobic feature leads to its poor solubility, limited bioavailability and high lipophilicity which restrict it from reaching the cancer sites effectively.
Cervical cancer is one of the most common female malignancies worldwide and over 90% of cases are associated with high-risk HPV infection. Current treatment involves surgery, radiotherapy and chemotherapy depending on the stages and cisplatin is considered the most common drug choice despite its cytotoxicity.
In This study, unconjugated liposomal encapsulated thymoquinone (TQ) and folate conjugated liposomal encapsulated TQ were prepared and they were used to treat HPV16+ CaSki, HPV- C33A and normal human keratinocyte HK cell lines.
The anti-proliferative effect of TQ and cisplatin was compared by determining their half-maximal inhibitory concentration (IC50) after 24 and 48 hours of drug exposure via performing MTT assay using HPV16+ CaSki, HPV- C33A and normal human keratinocyte (HK) cell lines. Results showed that TQ exhibited an anti-proliferative effect in a time and dose-dependent manner on the studied cancer cells but this effect was significantly reduced for the normal HK cells.
Liposomal encapsulated TQ (Lip-TQ) and folate-conjugated liposomal TQ (FA-Lip-TQ) were prepared and the encapsulation rates and physiochemical characteristics of prepared liposomal drugs were monitored over 17 weeks. Results showed that initial TQ encapsulation efficiency for both Lip-TQ and FA-Lip-TQ was 81% and 89% with their size ranging from 149.50nm±2.35 to 150.2nm±1.56 and 150.1nm±2.12 to 155.2nm±1.58 respectively. The zeta potential values demonstrated that both synthesised Lip-TQ and FA-Lip-TQ were neutral liposomes with PDI values lower than 0.2 indicating their homogeneity.
Furthermore, in order to enhance the specific targeting for cancer cells, a suitable surface tumour marker that is highly expressed on those cancer cells need to be identified. Folate receptors (FRs) have been well accepted as highly expressed receptors on cancer cells, therefore, they were evaluated on the three tested cell lines and the result showed that HPV positive CaSki cells expressed the highest levels of FRs among all three cell lines, followed by C33A cell line. FRs were scarcely expressed in HK cells.
The stability and TQ encapsulation efficiency from both Lip-TQ and FA-Lip-TQ were evaluated prior to their exposure to the cervical cancer cell lines. The anti-HPV effect of both free and liposomal encapsulated drugs (unconjugated and conjugated by FA) were investigated and results obtained demonstrated that FA-Lip-TQ was very effective in reducing the expression levels of HPV oncoproteins E6 and E7 in CaSki cells and in upregulating tumour suppressor proteins p53 and pRb comparing to free TQ and, Lip-TQ. For C33A cells Lip-TQ and FA-Lip-TQ were shown to be less effective in upregulating the tumour suppressor proteins compared to free TQ.
Also, FA-Lip-TQ showed more effectiveness in inducing cell apoptosis for CaSki cells than C33A cells. The proportion of early apoptotic CaSki cells after FA-Lip-TQ treatment was 37.7% which was significantly higher (p=0.0008) than the population of early apoptotic CaSki cells after Lip-TQ treatment (26.6%). However, the late apoptotic cells population did not follow the same trend. FA-Lip-TQ treated one was 7.18% which was less than those populations following Lip-TQ treatment (9.28%) (p=0.0005). As for the necrotic cell population, more was shown from Lip-TQ samples (4.82%) than FA-Lip-TQ treated one (2.98%) (p=0.002).
In the case of C33A cells, the population of early apoptotic cells (25.5%) after Lip-TQ treatment was higher than FA-Lip-TQ treated cells (22.7%) (p=0.0002), however, the late apoptotic cell population showed the opposite trend, in which more cells were shown from FA-Lip-TQ treated samples (17.7%) than Lip-TQ treated cells (13.5%) (p=0.0006). In addition, FA-Lip-TQ was shown to be more effective in upregulating both tumour suppressor proteins for both CaSki and C33A cell lines compared with free TQ and unconjugated Lip-TQ.
In conclusion, FA-Lip-TQ demonstrated a higher selectivity and efficiency in inducing apoptosis, downregulating HPV E6 and E7 oncoproteins and upregulating the tumour suppressor proteins p53 and pRb for HPV+ CaSki cancer cells than HPV- C33A cells. Therefore, using FA-conjugated liposomes to deliver TQ could play a crucial role in interfering with the life cycle of HPV viruses and the development of HPV associated cancers. |
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